bluuebunny/crossref_metadata_2025_split huggingface dataset:
Adenosine A1 receptor‐operated calcium entry in renal afferent arteriolar smooth muscle cells is dependent on postnatal maturation of canonical transient receptor potential isoform 3 channels
Authors: Peixoto‐Neves Dieniffer , Soni Hitesh , Buddington Randal K , Adebiyi Adebowale 
Published: November 2023

Adenosine, a regulator of cardiovascular development and renal function constricts renal afferent arterioles by activating its cognate A1 receptors (AA1Rs). Activation of AA1Rs elevates intracellular Ca2+ ([Ca2+]i) concentration in renal vascular smooth muscle cells, mechanisms of which are not fully resolved. Whether AA1R expression and function in preglomerular microvessels are dependent on postnatal kidney maturation are also unclear. Here, we show that selective activation of AA1R by 2‐chloro‐N6‐cyclopentyladenosine (CCPA) does not stimulate store‐operated Ca2+ entry in afferent arterioles isolated from infant pigs. However, CCPA‐induced [Ca2+]i elevation is dependent on phospholipase C, protein kinase C, and canonical transient receptor potential isoform 3 (TRPC3) channels. The basal [Ca2+]i concentration was unchanged in afferent arterioles isolated from newly farrowed (0 day) pigs when compared with their 20‐day old counterparts. Whereas, CCPA‐induced [Ca2+]i elevation was ~ 43% larger in afferent arterioles from 20‐day old pigs. AA1R protein expression levels in the kidneys and afferent arterioles were unaltered in 0‐day versus 20‐day old pigs. By contrast, TRPC3 channel protein expression was ~ 92 and 78% higher in 20‐day old pig kidneys and afferent arterioles, respectively. These data suggest that activation of AA1Rs elicits receptor‐operated Ca2+ entry in porcine afferent arterioles, an effect dependent on postnatal maturation of TRPC3 channels.Support or Funding InformationNIH R01 DK101668
Role of gap junctions in renal vascular conducted response
Authors: Moller Sophie 
Published: November 2023

The juxtaglomerular apparatus (JGA) is an essential structure in the regulation of renal function and embodies two major functions: tubuloglomerular feedback (TGF) and renin secretion. Both mechanisms function to regulate renal microcirculation, in addition to a plethora of systemic effects. The JGA is a unique structure to study intercellular communication. Intercellular connections, gap junctions (GJ), allow exchange of ions, nutrients, and small signaling molecules between neighboring cells. GJs consist of two connexons, one from each cell, with connexins (Cx) as their building blocks. Several Cx isoforms are expressed in the JGA [1], making the JGA a functional syncytium. Disruption of the signaling pathways in the JGA is associated with reduced TGF response, dysregulation of renin secretion, and hypertension [2,3]. The aim of the study was to determine if reduced intercellular communication affects the conduction of TGF‐induced vasoconstriction in the afferent arteriole.Experiments were performed using the isolated perfused juxtamedullary nephron preparation. Kidneys were isolated from wild type (WT) and knockout (KO) of both Cx40 and Cx45 mice. Kidneys were perfused with a Tyrodes buffer containing 5% BSA and an amino acid mixture, at pH 7.4. The inner renal surface contains a unique nephron population and allows access to the afferent arteriole. Local afferent vasoconstriction was induced by electrical pulse stimulation (300ms, 90V), administered via 2M NaCl filled microelectrode (0.5–0.8 MΩ) at the glomerular entrance. These electrical stimulations emulate the initiation of vascular TGF responses. Renal perfusion pressure was kept constant at 95 mmHg. Inner afferent arteriolar diameter was measured at the stimulation site (0 μm) and every 50 μm, at distances upstream (up to 450 μm), following a protocol consisting of a 5 min acclimation period, 30 sec baseline, 30 sec electrical pulse stimulation, and 30 sec recovery measurements. Responses between WT and KO of both Cx40 and Cx45 mice were compared.Preliminary data shows that electrical stimulation at the glomerular pole of the afferent arteriole reduces the diameter in all four groups at the local stimulation sites, with the vasoconstriction effect attenuated at upstream sites. A loss of vasoconstriction in response to stimulation is observed at 200 μm in Cx40 KO mice, and at 250 μm in Cx45 KO mice. In the WT mice, attenuation in the vasoconstriction is observed further upstream, at 450 μm in Cx40 WT mice. In the Cx45 WT mice no attenuation is observed at the measured distance of 300 μm.These results suggest that both Cx40 and Cx45 may play a significant role in the propagation of the afferent vasoconstriction elicited by electrical stimulation, and thus in the TGF signaling in the JGA.
A New View of Macula Densa Cell Biology
Authors: Shroff Urvi Nikhil , Perin Laura , Peti‐Peterdi Janos 
Published: November 2023

Macula densa (MD) cells of the juxtaglomerular apparatus (JGA) perform important, well‐characterized physiological regulatory functions including tubuloglomerular feedback‐mediated control of glomerular hemodynamics and renin release. Established chemical mediators synthesized and released from MD cells include ATP, adenosine, PGE2, and NO that are of non‐protein nature. However, the unique features of MD cell organelles, the apically located giant nuclei and the robust size of protein synthesis organelles at the cell base suggest that the MD is a massive protein factory. The present study aimed to characterize the basic MD cell biological processes and their regulatory mechanisms. Immunofluorescence localization of the key molecular players in DNA synthesis, cell cycle regulation, protein synthesis, processing, and vesicular release was performed in fixed mouse and human kidney sections. The high level and MD‐specific expression of the key cell cycle regulators Geminin and Wee1 suggested that MD cells are in cell cycle arrest in the G2/M phase. Staining for γ–tubulin revealed multiple centrosomes in some MD cells suggesting DNA endoreduplication (endocycling) and multiploidy. The robust and MD‐specific labeling of secreted angiogenic peptides (Ccn1, Pappa‐2, Gdf15) was localized in numerous small intracellular vesicles, suggesting the high rate of synthesis of these proteins and their release. The high level of MD‐specific expression of unique elements of the actomyosin motor complex (Myh10, Ablim1, Dctn2, Gsn) was also consistent with the presence of a well‐controlled vesicular release (exocytosis) machinery. In summary, MD cells have a unique cell biology and genetic program to power a robust synthetic and secretion machinery of various novel MD‐specific proteins that likely perform important functions in renal tubulo‐interstitial, vascular, and glomerular tissue remodeling.Support or Funding InformationNIH R01 64324, AHA, ADA
Altered expression of aquaporin water channels in a rat model of chronic diarrhea due to bile acid malabsorption
Authors: Yde Jonathan , Borg Johan , Fenton Robert A. , Moeller Hanne B. 
Published: November 2023

BackgroundBile acid malabsorption (BAM) is a condition where excess amounts of bile acids (BA) in the colon cause chronic watery diarrhea. BAM occur following resection or inflammation of the terminal ileum, cholecystectomy or as a primary condition, where the feedback regulation of bile acid (BA) synthesis is defective. The mechanisms by which BAs in the large intestine cause diarrhea are largely unknown. My aim is to determine if aquaporin water channels (AQPs) in the colonic epithelium are involved in the development of diarrhea during BAM.MethodsSix rats kept in metabolic cages were fed a diet containing 1% BA for 10 days as a model for BAM. Six other rats fed standard chow served as controls. Colonic epithelial cells were isolated by Ca2+‐chelation. Cells were processed for RNA and protein analyses. Colonic tissue was also prepared for histology.ResultsBA‐fed rats had greater feces dry weight and feces water content compared to rats fed standard chow. BA rats also had a higher water intake than controls. There were no differences in urine output or urine osmolality between groups. In contrast to controls, BA rats did not gain weight over time. The difference in weight was significant at the end of the experiment. Semi‐quantitative real‐time PCR of epithelial cells showed that organic solute transporter β (Slc51b) mRNA was increased in the colonic epithelium from the BA‐treated group compared to the control group indicating activation of the nuclear bile acid receptor (FXR). Aqp3 mRNA was increased in the distal colonic epithelium in the BA‐treated group, while mRNA for Aqp8 was increased in both the proximal and the distal colonic epithelium. mRNA for Aqp1, ‐4 and ‐7 was not different between groups. AQP3 was detected as a non‐glycosylated and a glycosylated form by western blotting. The glycosylated form was significantly reduced in the distal colonic epithelium of BA rats compared to controls. AQP8 was significantly increased in the distal colonic epithelium of BA rats compared to controls.PAS‐staining showed no clear differences in the histology of the colons from BA rats compared to colons from control rats. The expression pattern of AQP3 and ‐8 as shown with immunohistochemistry was not different between BA rats and controls.ConclusionIn a rat model of BAM we find increased expression of Aqp3 and Aqp8 mRNA and alterations of protein expression of AQP3 and ‐8 in the distal colon indicating a role for AQPs in the development of diarrhea during BAM.Support or Funding InformationSupport for my PhD‐study comes from the Faculty of Health's Research training supplement as well as from my main supervisor, Hanne Moeller, who received a Starting grant from Aarhus University Research Foundation, when she was hired as associate professor.
A Study on Mechanism and Potential Application of Novel Chalconoid Derivatives as Anti‐Secretory Therapy for Diarrheal Diseases
Authors: Yibcharoenporn Chamnan , Chavasiri Warinthorn , Chatsudthipong Varanuj , Muanprasat Chatchai 
Published: November 2023

Secretory diarrhea is one of major health problems that mainly results from Cl− secretion through apical Cl− channels including cystic fibrosis transmembrane regulator (CFTR) and calcium‐activated chloride channel (CaCC). Naturally derived chalcone (isoliquiritigenin or ISLQ) has previously been shown to inhibit CFTR with IC50 of about 20 μM without affecting CaCC. The aims of this study were to optimize chalconoid derivatives for their inhibitory effect against both CFTR and CaCC, and to explore their underlying mechanisms. The screening of chalconoid derivatives using short‐circuit measurements in T84 cells, a human intestinal epithelial cell line, identified an chalcone derivative CHAL‐025 that inhibited both CFTR and CaCC with higher potency than ISLQ (IC50 for CFTR 1–5 μM and IC50 for CaCC 3–10 μM). CHAL‐025 at concentrations up to 20 μM showed no cytotoxic effect on T84 cells. The inhibition of CFTR‐mediated chloride secretion was reversible and only observed when CHAL‐025 was added at the apical side. In addition, IC50 of CHAL‐025 in inhibiting CFTR did not change when basolateral membrane was permeabilized, suggesting that CHAL‐025 is apically active and the inhibitory effect of CHAL‐025 is insensitive to membrane potential. CFTR inhibitory potency of CHAL‐025 was not affected by pretreatment with inhibitors of protein kinase A (PKA), protein phosphatase (PP) and phosphodiesterase (PDE), indicating that mechanisms of CFTR inhibition by CHAL‐025 did not involve PKA, PP and PDE. Mechanism of CaCC inhibition by CHAL‐025 was also investigated and will be presented. Importantly, CHAL‐025 inhibited chloride secretion induced by bile acid across T84 monolayers, which is an experimental model mimicking bile acid‐induced diarrhea. In conclusion, the present study identified a chalcone derivative that is of potential utility in the treatment of secretory diarrheas resulted from both CFTR and CaCC overstimulation.Support or Funding InformationThis work was supported by grants BRG5980008 and DBG5980001 from the Thailand Research Fund and Mahidol University and by the Faculty of Science, Mahidol University and the Medical Scholars Program, Mahidol University.
A Screen for Potassium‐sensitivity in Yeast Reveals Endosomal Sorting Factors that Regulate the Renal Outer Medullary Potassium (ROMK) Channel
Authors: Mackie Timothy D. , Welling Paul A. , Subramanya Arohan R. , Brodsky Jeffrey L. 
Published: November 2023

The renal outer medullary potassium (ROMK) channel is a major route for potassium secretion from distal nephron principal cells in response to changes in dietary potassium intake. In addition, ROMK acts in tandem with the sodium‐potassium‐chloride cotransporter‐2 (NKCC2) to facilitate sodium reabsorption in the thick ascending limb. Genetic perturbations in ROMK cause an autosomal recessive salt‐wasting disorder in newborns known as Bartter Syndrome Type II, while these same mutations provide heterozygote individuals with resistance to developing hypertension. To date, ROMK anterograde trafficking through the secretory pathway and endocytosis from the plasma membrane in renal epithelia are well studied, however little is known about the factors that regulate either the post‐endocytic fate of ROMK or the fate of ROMK in the early secretory pathway. To rectify this deficiency in our understanding of how ROMK is regulated, we developed an assay utilizing a genetic screening technique in the budding yeast Saccharomyces cerevisiae. In brief, yeast were rendered sensitive to low potassium by deletion of the two native potassium transporters, Trk1 and Trk2. ROMK was then transformed into these cells, and crossed with a collection of 5,000 unique strains, each of which have a single ORF deleted. Strains that grow better on low potassium media are hypothesized to be deficient in negative regulators of ROMK. Several factors related to endosomal sorting were identified through this screen, including members of the class‐C core vacuole‐endosome tethering (CORVET) complex, which brings endocytic vesicles together to form early endosomes, as well as the endosomal complexes required for transport (ESCRT), which sort ubiquitinated cargo into multi‐vesicular bodies prior to degradation in the lysosome/vacuole. These findings were validated by serial dilution growth assays in independently constructed yeast strains. Next, these findings were validated in a more relevant model. Cell surface biotinylation of HEK293 cells treated with siRNA to knockdown human orthologues of screen hits indicated that CORVET and ESCRT also deplete ROMK from the plasma membrane in human cells. These genetic data lead us to propose that the plasma membrane population of ROMK is endocytosed and sorted to the lysosome for degradation in a CORVET and ESCRT‐dependent manner. In addition, hypothesis‐driven studies in yeast showed that ROMK is also partially regulated at the level of the endoplasmic reticulum (ER) via ER‐associated degradation (ERAD). Together, these findings enhance our understanding of the cellular itinerary of this critical ion channel and may lead to the development of novel therapies for patients with Bartter syndrome and other disorders of potassium balance.Support or Funding InformationThis work was supported by grants GM75061 and DK79307 to J.L.B from the National Institutes of Health
Effect of Diuretics and Gender on Kcnj16‐mediated Effects in Salt Sensitive Hypertension
Authors: Palygin Oleg , Levchenko Vladislav , Prudnikov Oleh , Nguyen Christine , Staruschenko Alexander 
Published: November 2023

Hypokalemia is a potentially life‐threatening imbalance characterized by a serum potassium concentration of less than 3.5mmol/L (severe hypokalemia < 2.5mmol/L) caused by a deficit in total body potassium stores induced by dietary, genetic or iatrogenic factors. Inwardly‐rectifying K+ channels Kir4.1 (Kcnj10) and Kir5.1 (Kcnj16) are highly expressed on the basolateral membrane of distal renal tubules and contribute to Na+ reabsorption and K+ secretion through the direct control of trans‐epithelial voltage. To define the importance of Kir5.1 in electrolyte balance and potassium homeostasis in the body, we generated a Kcnj16 knockout in Dahl salt‐sensitive (SS) rats (SSKcnj16−/−). SSKcnj16−/− rats fed a normal 0.4% NaCl diet (Dyets) had severe hypokalemia (3.8±0.03 vs 2.00±0.06, mmol/L; SS vs SSKcnj16−/− rats correspondingly, p<0.05, N≥9) and reduced blood pressure. Female rats had an even more profound change in blood potassium (3.5±0.04 vs 1.55±0.04 mmol/L; SS vs SSKcnj16−/− rats correspondingly, p<0.05, N≥11). Overall fractional excretion analyses further revealed that SSKcnj16−/− rats had a salt‐wasting phenotype with increased FENa+, FEK+, and FEMg2+. When fed a high salt diet (4% NaCl; Dyets) SSKcnj16−/− rats experienced 100% mortality triggered by diuresis‐natriuresis and a further drop of blood potassium level below 1.4mmol/L. To reduce mortality and preserve potassium wasting under high salt conditions, we supplemented the drinking water of the SSKcnj16−/− rats with inhibitors of sodium channels and transporters critical for maintenance of electrolyte homeostasis in the Thick Ascending Limb (TAL) (furosemide; 150mg/L), Distal Convoluted Tubule (DCT) (Hydrochlorothiazide (HCTZ) 75mg/L) and Cortical Collecting Duct (CCD) (benzamil 15mg/L) (N≥6 for males and females in each treatment group). Epithelial sodium channel (ENaC) inhibitor benzamil, but not other diuretics, rescued SSKcnj16−/− rats from mortality on the high salt diet, but removal of benzamil from the drinking water after two weeks of high salt treatment still resulted in mortality. Interestingly, treatment with HCTZ delayed mortality only in males and had no effect in females, emphasizing the role of the DCT segment specifically in male SSKcnj16−/− rats. Together, our data with diuretics targeting NKCC, NCC and ENaC revealed that only benzamil treatment rescued SSKcnj16−/− rats when challenged with a high salt diet, and furthermore we have demonstrated gender differences important in regulation of Kcnj16‐mediated effects in the kidney.Support or Funding InformationMedical College of Wisconsin Neuroscience Research Center/Advancing a Healthier Wisconsin pilot grant #9520217 (to OPa) and National Institutes of Health grants HL108880, HL122662 (to AS)
Cerebrovascular Responses to Trigeminal Nerve Stimulation in Humans
Authors: Ahmed Amar , Edwards Louisa , Braz Igor Dutra , Fisher James P 
Published: November 2023

The mammalian diving reflex, evoked by cold water face immersion, is characterized by peripheral vasoconstriction, an increase in blood pressure (BP) and a concomitant reduction in heart rate (HR). Trigeminal nerve stimulation (TGS) partly mediates these reflex induced cardiovascular adjustments, which are thought to conserve blood flow and oxygen for delivery to the brain and heart. We aimed to characterise the cerebrovascular responses to TGS under isocapnic conditions, and those evoked by generalized sympathetic activation during thermoreceptor stimulation with ice water hand immersion (cold pressor test; CPT).In eight healthy men (23±6 years) continuous recordings of middle cerebral artery velocity (MCA Vmean; transcranial Doppler), HR (ECG), mean BP (Finometer) and the partial pressure of end‐tidal carbon dioxide (PETCO2) were obtained. Cerebral vascular conductance index (CVCi) was calculated as; MCA Vmean/MAP. Participants underwent 3 minutes of CPT, with ice water (4°C) right hand immersion, and 3 minutes of TGS evoked by ice packs placed on the forehead and cheeks. Trials were randomized and separated by a 20‐minute resting period. Supplemental CO2 was added to the inspired air in order to maintain isocapnia throughout the TGS and CPT trials.During TGS a decline in heart rate was observed, while mean BP rose progressively (p<0.05). MCA Vmean did not change significantly with TGS (baseline; 56.9 ± 3.4 cm/s, minute 3; 56.2 ± 3.3 cm/s, p>0.05), while CVCi was reduced (baseline; 0.79 ± 0.50 cm/s, minute 3; 0.66 ± 0.47 cm/s, p<0.05). In contrast, CPT was associated with a transient increase in HR during the first minute, and a constant elevation in mean BP (p<0.05). MCA Vmean was significantly increased during CPT (baseline; 57.5 ± 2.9 cm/s, minute 3; 64.6 ± 3.6 cm/s; p<0.05) and CVCi decreased (baseline; 0.73 ± 0.41 cm/s, minute 2; 0.64 ± 0.38 cm/s, p<0.05). PETCO2 remained unchanged from baseline in both TGS and CPT trials.These preliminary findings suggest that cerebral perfusion is elevated during generalized sympathetic activation evoked by the CPT, whereas MCA Vmean remains unchanged during TGS under isocapnic conditions.Support or Funding InformationJPF is funded by the British Heart Foundation.
Does maximal isokinetic power at exercise intolerance approximate critical power?
Authors: Yong Stanley , Swisher Austin , Ferguson Carrie , Cannon Daniel T 
Published: November 2023

Tolerance to high‐intensity exercise is described by a hyperbolic power‐tolerable duration (P‐tLIM) relationship. The asymptote of the P‐tLIM relationship, termed critical power, demarcates sustainable from non‐sustainable exercise. However, characterizing this relationship is time consuming – typically 4 constant power exercise tests to intolerance are required. Alternatively, at the limit of tolerance to supra‐critical power exercise, a 3 min all‐out sprint approximates critical power (Murgatroyd et al., Eur J Appl Physiol 114:1863–74, 2014). We aimed to determine whether maximal isokinetic power (30 s in duration) measured immediately at the limit of tolerance approximates critical power.Ten participants (3 females, 7 males, 22±2 yr, 73±11 kg, VO2peak 3.62±0.87 L.min−1) completed constant supra‐critical power exercise tests to intolerance, immediately followed by a maximal isokinetic effort at 80 rpm for 30 s. Mean isokinetic power over the final 20 s was compared to the critical power asymptote determined from four variable‐duration constant power tests to intolerance. Mean bias and limits of agreement were calculated using the method of Bland & Altman.Critical power estimated from the P‐tLIM relationship was 165±54 W with 95% confidence interval of 157, 172 W. Of the four constant power tests used to define P‐tLIM, isokinetic power was not different to critical power only following the longest duration trial (p>0.05, 186±65 vs 165±54 W). However, mean bias and limits of agreement were 21 W and ±88 W, respectively. In the remaining trials, isokinetic power immediately following the limit of tolerance was greater than critical power (p<0.05).Isokinetic power measured immediately following the limit of tolerance consistently overestimated critical power. While not different to critical power, the closest estimation showed more than 20 W bias with limits of agreement nearly 12 times the span of the 95% confidence interval. Thus, brief maximal isokinetic power (30 s) immediately following the limit of tolerance does not approximate critical power.