bluuebunny/crossref_metadata_2025 huggingface dataset:
Important role for the mammalian‐target‐of‐rapamycin (mTOR) in the collecting duct as a determinant of transporter/channel profile and function
Authors: Brown Aaron L , Lee Hwal , Tiwari Swasti , Li Lijun , Ecelbarger Carolyn M.
Published: November 2023

mTOR, a serine‐threonine‐specific kinase, is known as a central energy integrator of a number of signaling pathways and may have a role in renal transport in the collecting duct (CD), including facilitating ENaC activation by insulin. To further elucidate the role of mTOR in the CD principal cell, we generated a CD‐principal‐cell‐select mTOR KO mouse by Cre‐lox recombination using aquaporin‐2 (AQP2) promoter to drive Cre‐recombinase. Mice were of normal appearance and body weight. Basal urine volume (24 hr) and kidney weights were not different. A benzamil‐sensitivity test showed that KO mice had significantly reduced urine chloride (Cl−) concentrations (mM), as well as, absolute excretion of both sodium (Na+) and Cl− (μmol/4 hours), as compared to the WT suggesting reduced ENaC activity. To further explore this finding, urine (24‐hr) was collected in male mice under normal (NS) and low‐Na+ (LS) diets (< 0.05% Na+) over the course of a week (n = 14 mice/genotype/diet). Urine Na+ concentrations under LS diet were higher in the KO on day 1, but were reduced to a similar level by the end of the week. Surprisingly, urine aldosterone excretion was slightly, but significantly, lower in the KO mice in the basal period and after 1 day LS, but rose to similar level as the WT mice by day 7. After a recovery week, mice were split into two groups and randomly assigned to receive one‐of‐the‐two diets for another week, then euthanized. Blood analyzed by iSTAT showed modest alkalosis in the KO, especially under normal Na+, i.e., slightly elevated pH, HCO3, base excess, and reduced pCO2 (all significant differences). Plasma Na+ was slightly lower in the KO under NS, but not different from WT under LS. No differences in plasma K+ or Cl− were found. Western blotting conducted on cortex homogenates showed mTORKO had significantly reduced β‐ and γ‐subunits of the epithelial sodium channel (ENaC), aquaporin‐2 (AQP2), and rhesus protein family b‐glycoprotein (Rhbg) relative to WT under both dietary conditions. In the inner medulla, AQP2 was about 50% lower in the KO mice under NS, but actually 20% higher than WT under LS. We predict this may represent compensation for down‐regulation in cortical CD. Overall, these results support a central role for CD mTOR in homeostatic modeling of the CD and expression of key transport proteins.

Support or Funding Information

Georgetown University Department of Medicine; CTSA Georgetown/Howard; Department of Biochemistry (AB)
The Lack of Urea Transporters, UT‐A1 and UT‐A3, Increases Nitric Oxide Accumulation to Dampen Medullary Sodium Reabsorption Through ENaC
Authors: Sun Michael A. , Rogers Richard T. , Redd Sara K. , Yue Qiang , Bao Hui Fang , Eaton Douglas C. , Blount Mitsi A.
Published: November 2023

While the role of urea in urine concentration is known, the effect of urea handling by the urea transporters, UT‐A1 and UT‐A3, on sodium balance remains elusive. Serum and urinary sodium is similar between wild‐type mice (WT) and UT‐A3 null (UT‐A3 KO) mice; however, mice lacking both UT‐A1 and UT‐A3 (UT‐A1/A3 KO) have significantly lower serum sodium and higher urinary sodium. Protein expression of renal sodium transporters (NHE3, NCC, NaATPase, NKCC2, ENaC) is unchanged among all three genotypes. WT, UT‐A3 KO and UT‐A1/A3 KO acutely respond to hydrochlorothiazide and furosemide; however, UT‐A1/A3 KO fail to show a diuretic or natriuretic response following amiloride administration indicating that ENaC activity is impaired. Interestingly, IMCD biotinylation showed that UT‐A1/A3 KO have more ENaC at the apical membrane than WT, and single channel analysis of ENaC in isolated, split‐open IMCD revealed that ENaC channel density and open probability is higher in UT‐A1/A3 KO than WT. UT‐A1/A3 KO excrete larger amounts of urinary nitric oxide (NO), a paracrine inhibitor of ENaC, and express 40‐fold more inner medullary nitric oxide synthase 1 (NOS1) mRNA than WT. Because endogenous NO is unstable, ENaC activity was reassessed in split‐open IMCD with the NO donor, PAPA NONOate, and now found to be almost completely abolished in UT‐A1/A3 KO. In summary, loss of both UT‐A1 and UT‐A3 (but not UT‐A3 alone) cause a salt‐wasting phenotype and an elevation in medullary NO production. NO inhibition of ENaC, despite elevated apical protein accumulation in UT‐A1/A3 KO IMCD, appears to be the main contributor to natriuresis in UT‐A1/A3 KO mice.
Reversible modulation of the proximal tubule apical endocytic pathway by fluid shear‐stress
Authors: Shipman Katherine E , Long Kimberly R , Rbaibi Youssef , Baty Catherine J , Weisz Ora A
Published: November 2023

Epithelial cells that line the proximal tubule (PT) of the kidney are responsible for the reabsorption of low molecular weight proteins and other small molecules from the glomerular ultrafiltrate. A limitation in understanding the regulation of receptor mediated apical endocytosis of filtered ligands has been the lack of a highly differentiated cell culture model for the proximal tubule. Because PT cells in vivo are continually exposed to flow, we have cultured cells under continuous fluid shear stress (FSS) to better recapitulate this environment. Opossum kidney cells were plated on permeable filter supports, and the following day were either exposed to orbital FSS or maintained under static conditions for an additional four days. Ultrastructural analysis revealed striking differentiation of the apical brush border and endocytic pathway in these cells, similar to that observed in PT cells in vivo. Endocytic capacity per cell, quantified based on uptake of fluorescently‐labeled albumin, was also dramatically increased in cells cultured under FSS. Endocytic capacity was rapidly decreased upon reducing FSS. Consistent with enhanced endocytosis, we found a significant increase in expression and a change in distribution of Rab11a, a marker of recycling endosomes, in cells cultured under FSS. Batch deconvolution of confocal images will be implemented prior to quantitation of colocalization of megalin, its ligands, and known components of the endocytic machinery to measure the effect of FSS on the apical endocytic pathway. We are currently working to identify changes in other trafficking proteins that will be used to construct a testable model for how the endocytic pathway is acutely regulated in differentiated PT cells.

Support or Funding Information

National Institutes of Health R01‐DK101484, R01‐DK100357, P30‐DK079307
Comparative proteomic analysis of kidney distal convoluted tubule and cortical collecting duct cells following long‐term hormonal stimulation
Authors: Wu Qi , Møller Hanne , Rosenbæk Lena Lindtoft , Pisitkun Trairak , Fenton Robert A.
Published: November 2023

The distal convoluted tubule (DCT) and the cortical collecting ducts (CCD) are portions of renal tubule that are partly responsible for maintaining the systemic concentrations of potassium, sodium, calcium and magnesium. Despite being structurally similar, DCT and CCD cells have different transport capabilities due to a variety of different membrane‐associated transport proteins. However, DCT and CCD cells appear to be modulated via the same hormones. The objective of this study was assess the differential response of DCT and CCD cells to long‐term exposure to the hormones vasopressin or angiotensin II, both of which modulate DCT and CCD cells differently. Mass spectrometry based quantitative proteomics was used to profile the differential proteome between DCT and CCD. Mouse kidney distal convoluted tubule cells (mpkDCT) were cultured in heavy SILAC medium (Lys+6, Arg+10) while cortical collecting duct cells (mpkCCD) were cultured in light SILAC medium (Lys+0, Arg+0). Four passages of labelled cells were used to generate four biological replicates for statistical analysis. In addition to control conditions, cells were also stimulated for 4 days with the vasopressin type II receptor agonist 1‐desamino‐8‐D‐arginine vasopressin (dDAVP, 1nM) or angiotensin II (ANGII, 1nM). Cells were harvested, equally pooled and subjected to offline high‐pH fractionation based two dimensional LC‐MS/MS analysis (Q‐Exactive). Identification and quantification of proteins was performed by MaxQuant. Proteins that had at least three ratios out of four biological replicates were defined as quantifiable proteins. Proteins that had at least three iBAQ values out of four biological replicates in light or heavy channel and zero iBAQ values in the other channel were defined as unique proteins in a particular cell type. Out of the some four thousand quantifiable proteins under basal, dDAVP and AngII conditions, 1101, 1566 and 1294 proteins passed the quantitative Benjamini‐Hochberg (BH) FDR 1% threshold, respectively. The mpkDCT or mpkCCD specific proteins were defined based on being an up‐regulated protein that passed the 1% BH FDR threshold in one cell type plus the unique proteins in this cell type. These 1025 mpkDCT specific proteins and 1211 mpkCCD specific proteins under the three conditions were subjected to further bioinformatics analyses including Panther and DAVID gene ontology analyses, E3 ligase and deubiquitinating enzyme identification, and kinase and transcription factor predictions, with the aim to identify cell specific proteins that define tubule‐specific biological processes. Preliminary data suggests that one specific gene CHIP in mpkCCD might be involved in the regulation of AQP2.

Support or Funding Information

Danish Council for Independent Research, Lundbeck Foundation
Nuclear Factor‐kB Mediates Zinc Deficiency‐Induced Sodium Chloride Cotransporter Upregulation in Mouse Distal Convoluted Tubular Cells
Authors: Naraine Meagan K , Li Mirandy S , Williams Jasmine , Mistry Monisha , Hoover Robert S , Williams Clintoria R
Published: November 2023

Zn2+ deficiency (ZnD), a common disorder in chronic kidney disease, contributes to hypertension. However, the mechanism is unknown. The Sodium Chloride Cotransporter (NCC) plays a critical role in blood pressure homeostasis. Recently, our lab found that ZnD leads to hyperactivation of NCC that was reversed with Zn2+ supplementation, suggesting a novel mechanism by which ZnD contributes to hypertension. Other studies have shown that Nuclear Factor‐kB (NFkB) is activated in ZnD. The purpose of this study was to determine the role NFkB plays in ZnD‐induced NCC upregulation. We hypothesize that ZnD activates NFkB, which leads to an upregulation of NCC. To investigate this mechanism, mouse distal convoluted tubular (mDCT‐15) cells were treated with the pharmacological agent Caffeic acid phenethyl ester (CAPE), a NFkB inhibitor, for 24 hours prior to inducing ZnD with the Zn2+ chelator TPEN for an additional 24 hours. NCC expression (via Western blot, qRT‐PCR, and immunofluorescence) and localization (via biotinylation) were examined. Compared to vehicle, TPEN increases NCC mRNA and protein expression. Additionally, NCC surface localization was enhanced. However, NFkB inhibition with CAPE prevents TPEN‐induced NCC upregulation. Based on these results, it is apparent that NFkB plays a role in ZnD‐induced NCC upregulation in mDCT‐15 cells. Understanding the mechanisms by which ZnD contributes to hypertension may have an important impact on the treatment of blood pressure dysregulation.

Support or Funding Information

Funding: NIH‐T32 (CRW); NIH‐ROI (RSH); VA‐MERIT (RSH); SUPERR (MKN)
NBCe1‐A Is Not The Only NBCe1 (SLC4A4) Isoform Expressed in the Mouse Kidney
Authors: Verlander Jill W. , Chen Chao , Lee Hyun‐Wook , Osis Gunars , Fang Lijuan , Chen AnPing , Holmes Heather L , Romero Michael F. , Weiner I. David
Published: November 2023

The A‐splice variant of Na+‐dependent, electrogenic bicarbonate transporter‐1 (SLC4A4, NBCe1‐A) is generally believed to be the only SLC4A4 isoform expressed in the mammalian proximal tubule, and to have critical roles in filtered bicarbonate reabsorption, proximal tubule ammonia metabolism and proximal tubule organic anion metabolism. The purpose of the current study was to examine expression of other splice variants in the mouse kidney. We used recently generated mice with TALEN‐generated deletion of A and D NBCe1 splice‐variants, but with intact expression of the B, C and E isoforms. RT‐PCR using primers specific to the B, C and E isoforms showed mRNA of non‐A,D splice variants was expressed in the kidney of mice with intact NBCe1‐A expression and with NBCe1‐A‐specific deletion. We then used an antibody that recognizes portions of NBCe1 present in all splice isoforms and examined immunolabel in the kidney of NBCe1‐A‐deletion mice. There was basolateral immunolabel in the proximal convoluted tubule in the cortex, and lesser intensity basolateral immunolabel in the proximal tubule straight tubule in both the cortex and outer medulla. Specificity of this antibody was demonstrated by identifying that mice with deletion of all splice variants had no detectable immunolabel. An antibody specific to the A‐splice variant showed basolateral immunolabel throughout the entire proximal tubule of mice with intact NBCe1‐A expression, but no detectable immunolabel in mice with NBCe1‐A‐specific deletion. Thus, an NBCe1 splice variant other than NBCe1‐A appears to be expressed in the mouse proximal tubule. In acid‐loaded NBCe1‐A‐deletion mice, immunolabel observed using the pan‐specific anti‐NBCe1 antibody showed increased immunolabel in the proximal straight tubule, particularly in the outer medulla. These studies show that splice variants of NBCe1 other than NBCe1‐A/D are present in the mouse kidney and that their expression appears to be increased with acid‐loading. We conclude that these splice variants may have important roles in proximal tubule filtered bicarbonate reabsorption, ammonia metabolism and organic anion metabolism.

Support or Funding Information

The studies were supported by funds from the NIH (R01–DK045788 to IDW, R01‐DK107798 to IDW and JWV, and R01‐EY017732 to MFR) and the Department of Veterans Affairs (1I01BX000818 to IDW).
Measuring intracellular protein concentration: why and how?
Authors: Rana Priyanka
Published: November 2023

In discussions of cell volume regulation, an assumption is often made that a cell can be characterized by its “natural” volume determined by some intrinsic set point. Although this may be true for stable and differentiated tissues, it is obviously wrong for actively dividing cell populations, where the volume doubles between divisions. In this case, it seems more natural to talk about a set point for protein concentration C, rather than for volume V. For example, the majority of data on regulatory volume increase or decrease have been collected within a brief period following the osmolarity change, presumably to avoid complications resulting from cell growth. By using the intensive property of protein/water concentration, rather than the extensive property of volume, this complication can be avoided. Another example comes from apoptosis. Apoptotic shrinkage can be caused by dehydration or by fragmentation. To distinguish between these two processes, one has to know both V and C. Thus, we have developed two simple methods based on light transmission microscopy, to measure these parameters.

Cell volume is measured by adding a strongly absorbing extracellular dye to the medium. If a transmission image is taken at a wavelength of maximal dye absorbance, cells appear brighter than the background in direct quantitative relationship to their thickness. This method gives accurate information on V and has been used extensively in our lab. At the same time, one can collect brightfield images away from the absorbance peak. There is a theory that relates transmission image intensity to the phase delay by means of the transport‐of‐intensity equation (TIE). We have further improved the accuracy of TIE by introducing additional calibration, so that protein concentration can now be expressed as C(g/ml) = 1.0·∑ϕ(radians·mm2)/V(mm3), where ϕ is the output of TIE and 1.0 is an empirical coefficient. This approach gives a new insight into cell adaptation to anisosmotic environment and into apoptotic shrinkage.
High sFlt‐1 Concentrations During Pregnancy Modulate the Ppara Promoter Methylation in the Fetal Liver
Authors: Stojanovska Violeta , Holwerda Kim , van der Graaf Anne Marijn , Boekschoten Mark , Faas Marijke , Scherjon Sicco , Plösch Torsten
Published: November 2023

Background

An adverse prenatal environment significantly increases the risk of chronic metabolic diseases in the offspring. The mechanisms underlying such programming are unclear but may involve epigenetic modifications that lead to altered fetal gene expression. It was demonstrated that poor maternal nutrition affects the DNA methylation status of important transcriptional factors in the liver. However, little attention has been given to other pathological events during pregnancy e.g. increased concentrations of soluble fms‐like tyrosine kinase‐1 (sFlt‐1). Our working hypothesis is that increased sFlt‐1 concentrations in the mother can influence the developmental plasticity and the phenotype of the offspring via resetting of DNA methylation marks.

Methods

Pregnant Sprague‐Dawley rats were injected with control or adenovirus overexpressing sFlt‐1 on gestational day (GD 8). Fetal plasma, body and organ weight were assessed on GD 19. Microarray analysis was performed to assess the offspring liver gene expressions. The candidate gene methylation status was verified by pyrosequencing.

Results

Plasma concentrations of sFlt‐1 peaked 48 h post injections in the treated animals, and the high concentrations were maintained until the end of the pregnancy. The fetal sFLt‐1 concentrations were increased and body weight and length were decreased in the sFlt‐1 group. There were no significant differences in the brain weight between the groups, but the livers were smaller in comparison to the respective control group. Microarray analysis revealed 623 differentially expressed genes in the fetal liver. The functional analysis indicated a significant effect on lipid metabolism and in particular fatty acid beta‐oxidation. Moreover, Ppara, a transcriptional factor and key regulator of hepatic lipid metabolism, was one of the most induced genes by the high sFlt‐1 concentrations present in the mother and in the offspring. In addition, we performed pyrosequencing, to confirm whether Ppara overexpression is accompanied by changes in the methylation of the respective promoter. The promoter region of Ppara revealed decreased methylation in the high sFlt‐1 group in comparison to the controls, which is in accordance to the observed gene overexpression.

Conclusion

Our data show that increased sFlt‐1 concentrations in the mother and the offspring lead to growth restriction, brain sparing and Ppara upregulation in the liver. This is accompanied by Ppara promoter hypomethylation. Overall this suggests that epigenetic mechanism may induce the altered phenotype of the offspring via high sFlt‐1 maternal concentrations.

Support or Funding Information

This work was supported by the Netherlands Organization for Health Research and Development (ZonMW, grant number 91211053).
Early Life Stress (ELS) induces Arginase 1 Gene Expression in the Renal Cortex of Adult Male Rats Under High Salt Diet
Authors: Obi Ijeoma E. , Pollock Jennifer S.
Published: November 2023

Early Life stress (ELS) increases cardiovascular disease risks. Human studies and animal models show that ELS is characterized by altered physiological function and immune dysregulation. Utilizing the rat model of ELS, maternal separation (MatSep), we previously showed that MatSep kidneys are in a pro‐inflammatory state. In this study, we aim to determine the effects of ELS on renal expression of inflammatory markers when challenged with a high salt diet for 7 days. We hypothesize that in response to a high salt challenge, rats would be more susceptible to a dysregulated inflammatory gene expression. MatSep involves separating the pups from the dam from postnatal day 2 (PD2) to PD14. Non‐separated pups were used as controls. At 12 weeks old, male control and MatSep rats were placed in metabolic cages under normal salt diet for 2 days and then switched to a high salt diet for 7 days. Food consumption, water intake and urine excretion were measured daily for the duration of the experiment. On day 7, kidney function was determined by creatinine clearance, sodium and potassium excretion was assessed using Easylyte plus analyzer, urine osmolality was measured using vapor pressure osmometer, and changes in inflammatory gene expression were determined in the renal cortex by quantitative real‐time polymerase chain reaction (qRT‐PCR). No significant differences were recorded for food intake, water intake, urine excretion, sodium and potassium excretion, and urine osmolality in MatSep rats compared to control rats. Likewise, creatinine clearance was similar in control and MatSep rats (control vs. MatSep; p>0.05; 3.53±0.41 mL/min vs. 3.48±0.19 mL/min). Interestingly, gene expression of arginase 1 (Arg1) was significantly increased in the renal cortex of MatSep rats compared to control rats (control vs. MatSep; fold change; 1±0.122 vs. 2.08±0.29; p=0.0122). Similar gene expression levels were observed for toll‐like receptor (TLR) 2 (control vs. MatSep; fold change; P>0.05; 1±0.31 vs. 1.40±0.341), TLR4 (1±0.44 vs. 1.44±0.49), and CD68 (1±0.46 vs. 2.14±0.86). No gene expression was detected for inducible nitric oxide synthase and interleukin 10. These findings suggest that high salt does not induce a difference in renal function of rats exposed to MatSep, although differences in Arg1 may reflect differences in renal injury.

Support or Funding Information

Funded by William Townsend Porter Pre‐doctoral Fellowship from the American Physiological Society to IEO and P01HL69999 to JSP
High fat diet exacerbates adipose tissue‐derived adipokines and hypertension in female C57BL/6 mice exposed to early life stress (ELS)
Authors: Loria Analia , Murphy Margaret O , Herald Joseph , Cohn Dianne
Published: November 2023

Early life stress (ELS) exposure has been recognized as an independent risk factor for obesity and cardiovascular disease. Maternal separation and early weaning (MSEW) during postnatal life show long‐lasting neuroendocrine and behavioral disturbances thereby is used as a mouse model of ELS. The goal of this study was to determine whether exposure to MSEW is associated with sex differences in adiposity, vascular function and blood pressure in response to high fat (HF) diet. MSEW was performed in C57BL/6 mice from postnatal days 2–16. Undisturbed litters served as control (C) group. Mice were placed on a low‐fat (LF, 10% kcal from fat) or HF (60% kcal from fat) diet for 16 weeks upon weaning. MSEW did not influence body weight gain in male and female mice fed a LF diet; however, MSEW increased fat mass with no changes in lean mass vs. C (p<0.05). In response to HF diet, despite significant increases in fat mass in both sexes, only female MSEW displayed higher levels of adiposity vs. C (44±2 vs. 31±2 %, p<0.05). In addition, MSEW exacerbated 24 hour mean arterial pressure (MAP) in both sexes; however, this increase in MAP was significantly greater in female (MSEW= 115.0±0.9 mmHg and C= 106.6±1.6 mmHg) than in male (MSEW= 121.2±1.3 mmHg and C=117.3±0.4 mmHg) mice (p sex<0.05, p MSEW p<0.05, p interaction <0.05). Yet, heart rate was increased only in male MSEW vs. C (p<0.05). Vascular reactivity assessed by wire myography showed increased serotonin‐induced vasoconstriction in female MSEW mice vs. C (73±15 vs. 36±3 %maximal constriction, p<0.05) but not in male MSEW mice vs. C (59±8 vs. 51±3 %maximal constriction, p<0.05). Plasma metabolic profile revealed that female, but not male, MSEW mice display hyperinsulinemia (1.3±0.1 vs. 0.5±0.1 ng/ml, p<0.05), hyperleptinemia (37.4±1.9 vs. 26.5±1.4 ng/ml, p<0.05), higher levels of plasma resistin (8.1±0.4 vs. 5.7±0.5 ng/ml, p<0.05) and PAI‐1 (1.7±0.2 vs. 0.5±0.0 ng/ml, p<0.05). Taken together, these data indicate that female mice exposed to MSEW are particularly susceptible to display fat expansion thereby increasing fat‐derived adipokines involved in vascular dysfunction and hypertension in response to HF diet.

Support or Funding Information

HL111354
Back to the Beach: Adaptations that Enabled Terrestrial Mammals to Return to the Water
Authors: Reidenberg Joy S.
Published: November 2023

Modern aquatic mammals are extremely diverse. This includes classic marine mammals such as cetaceans (whales, including dolphins and porpoises) and sirenians (manatees, dugongs), but also many amphibious mammals that spend some time on land (e.g., seals, otters, hippos, polar bears). Aquatic mammals retain terrestrial‐based systems that have been vastly modified for existence in water, but this alone does not make them extreme. However, within this group are some notable extremists: the cetaceans. They have the strangest anatomical aquatic adaptations – the ones with the “‐est” adjectives – enabling the deepest dive, slowest pulse, longest apnea, shortest exhalation, largest nose, narrowest mandible, strongest suction, biggest hyoid, widest mouth, stretchiest gular region, hairiest moustache, loudest sound, lowest song, longest tooth, highest vocalization, fattiest ear, tiniest external auditory meatus, densest tympanic bulla, curliest tongue, smoothest skin, stiffest cervical vertebrae, bumpiest forelimb, heaviest testes, and fastest copulation. For example, the sperm whale can dive to the bottom of the ocean and hold its breath for nearly an hour. Its head that is nearly one‐third of its body length, and is comprised of an enormous nose modified for sound production. Its mandible may plow the sea floor, and the hyoid and tongue create suction to engulf prey. The blue whale is the biggest mammal that ever existed, and has the widest mouth for engulfing prey. It has the largest larynx capable of making the loudest and lowest vocalizations. The narwhal has an elongated tooth that is likely the basis for the legend of the unicorn's horn. The dolphin uses modified nostrils to generate ultra high frequency sounds for echolocation, and uses acoustic fats as pinnae to receive sounds. A whale calf can curl the tongue into a straw for channeling milk when nursing underwater. Whale skin is very smooth enabling hydrodynamic water flow, and compressed/fused cervical vertebrae eliminate friction from lateral head deviations. The elongated flippers of humpback whales have bumps on the leading edge that increase efficiency. The porpoise is the smallest whale but has the largest testes and longest penis that can be rapidly extruded for the quickest mating. These amazing anatomical aquatic adaptations make cetaceans one of the most extreme groups of animals on Earth.
Integration of biomechanics and mechanobiology to predict chronic ventricular wall stress, thickness, and contractility
Authors: Fuertes Wesley W. , Shao Steven , Mohiuddin Hanifa S. , Lee Hyunjin , Dalal Fazal A. , Quick Christopher M. , Stewart Randolph H.
Published: November 2023

The ability of investigators to relate indices of cardiac function to structure, mechanical properties, and adaptive processes of ventricles suffers from isolation of complementary fields of study. On one hand, the field of biomechanics has clarified how ventricular wall stress (WS) varies with changes in contractility and wall thickness (WT). On the other, the less‐established field of mechanobiology has focused on how ventricular wall thickness and contractility vary with chronic alterations in ventricular wall stress. The purpose of the present work was to create a computational model that integrates biomechanics and mechanobiology to predict chronic ventricular wall stress, wall thickness and contractility. To capture fundamental behavior, we made six simplifying assumptions to mathematically model the left ventricle: cylindrical geometry, LaPlace's Law, time‐varying elastance, regulation of systemic arterial pressure, and end‐systolic elastance proportional to wall thickness. Most importantly, we made the critical assumption that wall thickness increases linearly in response to chronic wall stress. The result is a simultaneous prediction of wall stress, wall thickness, and end‐systolic elastance. This model not only predicts observed ventricular hypertrophy in response to elevated systemic pressure, but also reveals the central role of the sensitivity of the adaptive response (i.e., dWT/dWS) in modulating cardiac function, structure, and mechanical properties.
Relating preload recruitable stroke work to parameters characterizing systolic and diastolic cardiac function
Authors: Stoute Ian M. , Dekowski Lindsey P. , Le Kenneth K. , Dongaonkar Ranjeet M. , Quick Christopher M.
Published: November 2023

Preload recruitable stroke work (PRSW) is a fundamental characterization of cardiac function that is purportedly independent of preload and afterload. It is typically expressed as an empirical relationship between stroke work (SW) and end‐diastolic volume (Ved). This relationship has been reported to be strikingly linear over large ranges of end‐diastolic volumes in multiple mammalian species. Although the slope and intercept of the PRSW relationship shift significantly with different heart failure phenotypes, there is currently no means to predict a priori how changes in contractility, diastolic stiffness, or unstressed volume will affect the PRSW relationship. Therefore, the purpose of the present work was to develop an algebraic formula for the PRSW relationship in terms of standard parameters characterizing systolic and diastolic function. Four simple assumptions were made. First, the end‐systolic pressure‐volume relationship was assumed to be linear, characterized by end‐systolic elastance (Ees) and end‐systolic unstressed volume. Second, the end‐diastolic pressure was assumed to be an exponential function of end‐diastolic volume, characterized by parameters describing diastolic stiffness. Third, mean systemic arterial pressure (Psa) was assumed to be regulated. Fourth, mean systemic arterial pressure was assumed to approximate end‐systolic pressure. Solving for SW as a function of Ved results in a nearly linear relationship. The PRSW relationship was linearized using a Taylor series approximation, which yielded a simple formulas for the slope and intercept of the PRSW relationship in terms of Ees and diastolic stiffness. In addition, both the slope and intercept were found to be sensitive to Psa. This linear approximation provides students, physiologists, and clinical investigators with a novel tool that relates cardiac mechanical properties to cardiac function.
Algebraic formula predicting interstitial fluid pressure homeostasis from the interaction of microvascular filtration and lymphatic transport
Authors: Hasnain Syed Q. , Villanueva Mariana S. , Harris Anson Q. , Newman Courtney A. , Conley Megan C. , Nguyen Trisha B. , Quick Christopher M.
Published: November 2023

Interstitial fluid pressure remains relatively low despite fairly large increases in capillary and lymphatic outlet pressures. This maintenance of interstitial fluid pressure imposes competing requirements on lymphatic function. Edema results when lymph flow fails to adequately increase when interstitial fluid pressure rises, or when lymph flow fails to stay constant when lymphatic outlet pressure rises. This critical insight has not been incorporated in mathematical models that predict interstitial fluid pressures arising from the interaction of microvascular filtration and lymphatic transport. Although critical approximations made it possible to predict interstitial fluid pressure with a general algebraic formula, the assumption that lymph flow is equally sensitive to interstitial and outlet pressures neglects a fundamental homeostatic mechanism. Therefore, the purpose of the present work was to develop an algebraic formula predicting interstitial fluid pressure homeostasis that incorporates a differential sensitivity of lymph flow to interstitial and outlet pressures. A new model was constructed using three simple assumptions: 1) equilibrium is established when microvascular filtration into the interstitium equals lymph flow out of the interstitium, 2) microvascular filtration is governed by the Starling‐Landis equation, and 3) lymphatic flow increases linearly with interstitial fluid pressure and decreases linearly with lymphatic outlet pressure. Interstitial fluid pressure was solved algebraically as a function of capillary and lymphatic outlet pressures, as well as the microvascular filtration coefficient, protein reflection coefficient, and two new empirical parameters characterizing lymphatic function. Adding complexity to the model reproduces interstitial fluid pressure homeostasis with perturbations in capillary or lymphatic outlet pressures. Nonetheless, because interstitial fluid pressure is expressed as a simple algebraic formula, the results retain generality beyond a particular assumed set of parameters, make explicit how parameters interact synergistically, and enable new methods to derive values of critical parameters from experimental data.
Corticotropin‐Releasing Hormone Regulates Spontaneous Excitatory Postsynaptic Currents on the Nucleus of the Solitary Tract
Authors: Jia Shuping , Mifflin Steve
Published: November 2023

Corticotropin‐releasing hormone (CRH) plays a critical role in responses to stress. Two major subtypes of CRH receptors (CRHRs), type 1 receptors (CRH1Rs) and CRH type 2 receptors (CRH2Rs), have been identified in the medial nucleus tractus solitarius (mNTS). The goal of this study was to characterize whether CRH alters glutamatergic transmission to NTS neurons receiving arterial chemoreceptor afferent inputs. Whole‐cell recordings of NTS second‐order neurons identified by DiA labeling of carotid bodies were obtained in caudal NTS slices (250μM thick). Slices were incubated with 100nM CRH in aCSF for 20 minutes and spontaneous miniature excitatory postsynaptic currents (mEPSCs) were recorded under voltage clamp 1–3 hours later. Control slices were treated with aCSF alone. Pre‐treatment with CRH (100nM) significantly increased mEPSCs amplitude (p<0.01) from 20.7 ± 1.0 pA to 25.1 ± 1.1 pA, with no change in frequency (control: 2.2 ± 0.2 Hz; CRH: 3.1 ± 0.4 Hz, p>0.05). Pretreatment with the CRH antagonist astressin (100nM–1μM) for 10 minutes and then in combination with CRH for another 20 minutes attenuated the CRH‐induced increase in mEPSC amplitude to 19.3 ±1.3 pA (p<0.01). mEPSCs observed following application of CRH were abolished by the non‐NMDA glutamate receptor antagonist CNQX (20μM). These studies indicate that CRH has a direct effect on NTS neurons through postsynaptic CRHRs.

Support or Funding Information

This work supported by PO1 HL88052
Impact of Decentralization on Cholinergic Neurotransmission in Neurons of Mouse Major Pelvic Ganglia
Authors: Kyi Cindy , Garcia Virginia , Schulz David J
Published: November 2023

Major pelvic ganglia (MPG) of mouse contains postganglionic neurons innervating urinary bladder, the genitals and distal colon. Pelvic ganglia receive cholinergic parasympathetic inputs from the preganglionic neurons in the sacral cord through pelvic nerve, and sympathetic inputs from those in the lumbosacral cord through hypogastric nerve. The axons from the pelvic ganglion synapse onto postganglionic neurons on the target organ, releasing acetylcholine from parasympathetic axons and noradrenaline from sympathetic axons.

Damage to the pelvic nerve due to complications from surgeries carried out in the pelvic area often results in micturition and erectile dysfunctions. The goal of this project is to understand the impact of loss of neural input on nicotinic cholinergic transmission at the major pelvic ganglia at acute and chronic stages.

We performed unilateral decentralizations by transecting pelvic and hypogastric nerves on the right side of the mouse before their point of entry to the ganglion. The left side was left intact. We employed molecular and electrophysiology techniques on sham‐surgical control and decentralized adult male Swiss Webster mice. We measured the mRNA abundance of α2, α3, α4, α5, α6, α7, β2 and β4 nicotinic receptor subunits in whole MPGs using quantitative PCR (qPCR). We then measured synaptic transmission at MPG neurons using both current and voltage clamp methods. We measured synaptic strengths by recording excitatory postsynaptic currents (EPSCs) in response to pressure ejections of acetylcholine (ACh).

Our results indicate that loss of input to the MPG causes increased in synaptic strength of cholinergic neurons in both acute stage (24hours) and chronic stage (28 days) post‐injury. The increased levels were more pronounced in contralateral (intact) ganglia of the decentralized animals compared to ipsilateral (transected) ganglia at both acute and chronic stages. At 28 days post injury, the synaptic strength seems to be decreasing towards the sham‐control levels and the significant difference disappears for the contralateral ganglia. The increase in synaptic strength in decentralized ganglia were also accompanied by significant increase in mRNA transcript expression for α3 and β4 subunits. Physiological experiments involving subunit specific nicotinic acetylcholine receptor blockers suggest that α3 and β4 subunits seem to be the major subunits involved in nicotinic transmission at the mouse MPG. Hence, our study suggests that loss of input to mouse MPG results in increased in synaptic transmission of the MPG neurons and this increased level is also reflected at the level of mRNA transcript numbers for the major subunits involved in the synaptic transmission.

Support or Funding Information

This project was supported by funding from Missouri Spinal Cord Injury Research Program and Craig H. Neilsen Foundation.
Activation of N‐methyl‐D‐aspartate Receptor Inhibits Enkephalinergic Neurons in Mouse Spinal Superficial Dorsal Horn
Authors: Kato Eiko , Takemura Yu , Fukushima Teruyuki , Hori Yuuichi
Published: November 2023

Enkephalin (Enk)‐containing neurons are distributed at high concentration in the spinal cord superficial dorsal horn (SDH), where they play an important role in the transmission and modulation of nociceptive information. Recently, we performed physiological characterization of Enk‐containing neurons identified by green fluorescent protein fluorescence expressed under the control of the preproenkephalin gene promoter. We observed that Enk‐containing neurons in the SDH express the N‐methyl‐D‐aspartate receptor (NMDAR) 2B (NR2B) subunit and large conductance calcium activated potassium (BK) channels. In the present experiments, we investigated the involvement of NMDAR and BK channels in the regulation of the activity of Enk‐containing neurons in the SDH.

The experiments were performed in 5‐ to 8‐week‐old male ICR mice. Under ketamine/xylazine anesthesia, the lumbar spinal cord was dissected, and transverse slices were prepared. Tight‐seal whole‐cell recordings were obtained from neurons in the SDH. L‐glutamate (Glu) was applied by pressure ejection through a glass pipette placed near the recorded neurons. After the electrophysiological experiments, the recorded neurons were sampled, and single cell real‐time RT‐PCR was performed to analyze the expression profile of genes of interest in single SDH neurons. In some experiments, the left sciatic nerve was partially ligated, and mechanical allodynia was assessed by von Frey test.

Puff‐applied Glu evoked an inward current at a holding potential of −70 mV. Depolarizing the holding potential to 0 mV, outward current of long duration appeared subsequent to initial inward current. The NMDAR antagonist AP‐V abolished the outward current. The NR2B‐selective NMDAR antagonist ifenprodil reduced the outward current. The outward current was also abolished by the selective BK channels antagonist iberiotoxin. Single cell real‐time RT‐PCR analysis revealed that the neurons generating the outward current showed a significant tendency to express BK channels α‐subunit mRNA, which is consistent with the pharmacological results of a BK channels antagonist. Furthermore, single SDH neurons expressing the preproenkephalin mRNA also expressed the BK channel α‐subunit, NR1, and NR2B subunit mRNAs. The expression of NR2B increased significantly in sciatic nerve‐ligated mice.

Our results suggest that Ca2+ influx through NMDAR may activate BK channels and thus inhibit Enk‐containing neurons in the SDH. NMDAR‐mediated inhibition of Enk‐containing neurons is speculated to be one of the possible cellular mechanisms of chronic pain induced by peripheral nerve injury.
OVERWEIGHT IS ASSOCIATED WITH EXACERBATED IMPAIRMENT ON HEART RATE VARIABILITY IN OFFSPRING OF HYPERTENSIVE PARENTS
Authors: Viana Ariane , Nascimento Mário Cesar , de Paiva Júlia , dos Santos Fernando , Consolim‐Colombo Fernanda Marciano , Irigoyen Maria Cláudia , De Angelis Kátia
Published: November 2023

Considering the high prevalence of hypertension worldwide and the changes in lifestyle, which has been associated with the increase in the prevalence of overweight and obesity, it is necessary to study the mechanisms involved in the onset of such dysfunctions. The impairment on heart rate variability (HRV) has been associated with the onset of cardiovascular diseases and mortality. RedIn this sense, the objective of the present study is to evaluate cardiovascular and metabolic parameters and HRV in overweight individuals with and without family history of hypertension. This is a cross‐sectional study involving overweight sedentary men (24.9 ± 4.60 years old) with (OHT, n = 14) or without (ONT n = 10) familial history of hypertension. The body mass index (BMI) and skinfolds were evaluated. Blood lipid profile, fasting glucose and glycated hemoglobin (Hb1Ac) were analyzed. The blood pressure (BP) was measured by the auscultatory method. The RR interval was recorded during a 20 minute period at rest using a frequencimeter (Polar® RS800 model). The HRV was evaluated in the time and frequency domain. Both groups presented overweight (OHT: 30.4 ± 3.8kg/m2 and ONT: 27.7 ± 2.4 kg/m2) and elevated percent of fat mass (OHT: 34.7 ± 11.5% and ONT: 29.9 ± 9.7%). Lipid and glucose profiles were in normal range in both groups; however, the OHT group presented higher HB1Ac when compared to ONT group (5.4 ± 0.38 vs. 4.9 ± 0.32%, respectively). A negative correlation was obtained in the OHT group with respect to HDL and BMI (r2 = 0.81, p = 0.0028). Systolic (OHT: 121.1 ± 11.2 vs. ONT: 119.6 ± 5.9 mmHg) and diastolic BP (OHT: 75.5 ± 9.6 vs. ONT 77.7 ± 7.5mmHg) were similar between groups. We observed a decrease of the cardiac parasympathetic modulation (HF nu = 30.5 ± 7.2 vs. 47.43 ± 9.2%), an increase in cardiac sympathetic modulation (LF nu = 69.5 ± 7.2 vs. 52.5 ± 9.2%) and higher cardiac sympathovagal balance (3.73 ± 1.8 vs. 1.74 ± 0.75) in the OHT vs. ONT group. We conclude that family history of hypertension is associated with early metabolic and HRV dysfunctions in overweight individuals, suggesting higher predisposition to hypertension onset in this population.

Support or Funding Information

UNINOVE, CAPES‐PROSUP, CNPq.
The Effect of Transcranial Magnetic Stimulation on the Autonomic Nervous System in Pediatric Stroke
Authors: Chantigian Daniel , Rich Tonya , Chen Chao‐Ying , Lixandrao Maira , Gillick Bernadette , Keller‐Ross Manda
Published: November 2023

Transcranial Magnetic Stimulation (TMS) is used to study neurodevelopmental disorders including pediatric hemiparesis secondary to perinatal stroke and periventricular leukomalacia. Case reports of vasovagal syncope with TMS in adults and children suggest that TMS may have an effect on the autonomic nervous system (ANS); however, the effect of TMS on the ANS in children with stroke is unknown.

Objective

Determine blood pressure (BP) and heart rate (HR) responses to single‐pulse TMS in children with perinatal stroke and periventricular leukomalacia resulting in hemiparesis.

Methods

As a part of a larger study, autonomic data was recorded from 16 participants (9 females, 13 ± 4 yrs, mean ± SD) at rest and during resting motor threshold (RMT) testing of the primary motor cortex. RMT testing occurred for 15 ± 11 minutes. HR was continuously recorded via a 3‐lead electrocardiogram and BP was continuously recorded via a finger plethysmography device (AD Instruments, Colorado Springs, CO). Eight participants were excluded from the BP analysis because of unreliable data due to spasticity (n=5) and a lack of correct finger cuff size (n=3) in the monitoring hand. Autonomic data recorded during the first five stimulations were averaged and defined as the beginning of the stimulation session, while data recorded during the last five stimulations were averaged and defined as the end of the stimulation session.

Results

Average HR was similar at rest (85 ± 12 bpm) compared with the beginning of the stimulation session (82 ± 12 bpm, p = 0.06) and the end of the stimulation session (82 ± 9 bpm, p = 0.06). Average mean arterial pressure (MAP) was also similar at rest (79 ± 5 mmHg) compared with the beginning of the stimulation session (83 ± 7 mmHg, p = 0.29) and the end of the stimulation session (87 ± 18 mmHg, p = 0.24). Individual responses to TMS administration were variable and ranged from −17 to + 5 bpm for HR and from − 9 to + 32 mmHg for MAP.

Conclusion

Although on average TMS did not alter HR and BP in children with stroke, variations in individual responses to TMS were observed, suggesting that the ANS in some individuals might be more responsive to TMS administration than others. It is unclear if the observed changes in individual responses are a direct result of the stimulation or due to anticipation of receiving TMS (e.g. anxiety or stress). Because of the variations in individual HR and BP responses to TMS administration, monitoring the ANS may be important for identifying individuals that are at risk for syncopal events.