bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Human Neural Progenitor Cells and Neurons Differentiated from Induced Pluripotent Stem Cells
Authors: Apáti Ágota , Berecz Tünde , Szabó Eszter , Orbán Tamás István , Homolya László , Sarkadi Balázs , Réthelyi János
Published: June 2021

Most studies focusing on neural development and the mechanism of neurodevelopmental disorders are based on animal (rodent or avian) models or post mortem human samples. Thus, the observations derived from these studies cannot be directly translated into mechanistic insights of human neural development and regeneration. Human pluripotent stem cells, which can potentially be differentiated into any cell types of the body, offer a new perspective to overcome this limitation. In the present study, we differentiated induced pluripotent stem cell lines toward the neural lineage. Using a multi‐step, directed differentiation protocol, specific human neural progenitor cells (NPCs) were generated which are committed to produce PROX‐1‐positive granule cells of dentate gyrus of the hippocampus. These NPCs were characterized in terms of morphology, expression of specific cellular markers, growth capacity, and capability of producing mature neurons. Next we investigated the calcium signals in NPCs and PROX1‐positive hippocampal neurons, expressing GCaMP6fast calcium indicator protein or loaded with Fluo4 calcium sensitive dye, in parallel experiments. We demonstrated the functional presence of several receptors, and also the lack of functioning sodium channels in NPCs, and spontaneous Ca2+ gradients in neurons by both methods. We found that spontaneous calcium gradients and specific, ligand induced calcium signals were not significantly altered in the GCaMP6f expressing cells, suggesting the applicability of GCaMP6f for the investigation of fast calcium signals in neural cultures. We conclude that the characteristics of both the spontaneous and ligand‐induced Ca2+‐signals, as well as their pharmacological modulation can be successfully examined in these human model cells by fluorescence imaging.

Support or Funding Information

This study is funded by the National Brain Research Program (NAP) of Hungary (grant numbers: KTIA_NAP_13‐1‐2013‐0001 to LH and KTIA_NAP_13‐2014‐0011 to JR), and the Momentum Program of the Hungarian Academy of Sciences (LP2012‐025 to LH).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Immunomodulatory Effects of Rhesus Monkey Bone Marrow‐derived Mesenchymal Stem Cells in Serum‐free Conditions
Authors: Liu Fei , Li Yuanmin , Zhang Jie , Lu Yanrong , Cheng Jingqiu
Published: June 2021

Background

Mesenchymal stem cells (MSCs) has generated tremendous interest for treating various diseases due to the self‐renewal and differentiation. The great majority studies have indicated the immunoregulatory capability of MSCs, however, most researches are conducted based on the fetal bovine serum (FBS), meanwhile, commercial serum free media are not the promising candidates for cell therapy because of the confidentially uncertain composition. We aimed to develop a new serum free medium (SFM) suited for MSCs and evaluate the biological characteristics and the immunomodulatory potential of MSCs.

Methods

We established a complete chemical serum free media to support the proliferation and expansion of rhesus monkey bone marrow‐derived mesenchymal stem cells (rBMSCs), cells harvested through bone marrow aspiration from nine healthy rhesus monkeys. Passage 0 rBMSCs were cultured in serum containing media (SCM), following cell culture performed with serum free media and serum containing media respectively until P3. Growth kinetics, characterization, flow cytometry analysis, and immunosupressive abilities were evaluated compared with SCM. Moreover, RNA sequencing was used to evaluate the differentiated expressed genes (DEGs) related to immunization in two culture conditions.

Results

Compared to SCM, cells cultured in SFM exhibited better biological characteristics in the proliferation and immunnosupressive abilities. Both of cells showed similar immunophenotype for CD29, CD105, HLA‐DR, CD34, CD45. For RNA‐seq, five high correlated DEGs were enriched in immune system, only CXCL8 was down‐regulated (8 fold changes) acting as a pro‐inflammatory chemokine, which indicated that rBMSCs cultured in SFM had lower immunogenicity.

Conclusions

rBMSCs are feasible to be expanded safely in SFM and maintain the biological characteristics and immunosuppression functions in vitro. Further, this provides a possible cell culture methods for the clinical cell therapy requiring non‐animal derived demands, and improves the immunoregulatory effects of rBMSCs simultaneously, which could contribute to clinical therapies.

Support or Funding Information

This research was supported by Chengdu Giant Panda Breeding Research Foundation (2016cdspt001). Project name: Rhesus Monkey Mesenchymal Stem Cells Biological Study and Resource Pool Establishment

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protective Role of UCP2 against Oxidative Stress through Survival Signaling Pathway in Acute Epilepsy induced by Pilocarpine in Rats
Authors: Dutra Marina RH. , Feliciano Regiane Santos , Santana Eduardo T. , Jacinto Kalil R. , Gouveia Telma R. , Serra Andrey J. , Naffah‐Mazzacoratti Maria G. , Silva Jose A.
Published: June 2021

Neuroprotection is a desirable process in many human neurological disorders, yet complexes mechanisms involved in this field are not completely understood. The pilocarpine epilepsy model causes potent, seizure‐induced excitotoxicity cell death, and mitochondria impairment is also observed in this model. The present study was designed to verify neuroprotection of UCP2 against hippocampal epilepsy, using Real‐time PCR, immunohistochemistry, Western blot and RNA silencing. Herein, we demonstrated that rats subjected to an animal model of epilepsy induced by pilocarpine had increased levels of UCP2 mRNA expression in the acute phase. Additionally, the brains of rats injected with pilocarpine showed increased pro‐inflammatory makers mRNA expression. Moreover, these rats presented augmented p‐AKT expression only in the acute phase of the protocol, concomitantly with UCP2 mRNA augmentation. Rats treated with pilocarpine also presented an increase in oxidative stress, marked by damage caused by lipid peroxidation, higher levels of protein carbonyl, and a decrease in the levels of antioxidant enzymes, SOD and catalase. On the other hand, when pilocarpine‐treated rats received ASO therapy for UCP2, the number of seizures observed in the acute phase was increased compared to pilocarpine only injected rats. UCP2 silencing were able to worsening the oxidative stress parameters and diminishing p‐AKT expression. In conclusion, our study demonstrated that UCP2 might have neuroprotection after pilocarpine insult in hippocampi of rats.

Support or Funding Information

The authors wish to thank the CNPQ (308067/2014‐2) and FAPESP, PRONEX, Instituto Nacional de Neurociência Translacional, (INNT‐MCT), and CAPES.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Different Effects of Photobiomodulation Therapy on Cultured Human Chondrocytes
Authors: Zhang Junlin , Guffey Stephen , Motts Susan D. , Stroup Kyle J.
Published: June 2021

Introduction

The purpose of this study was to investigate the effects of photobiomodulation therapy (PBMT) with different wavelengths and fluences on the cell viability of cultured human chondrocytes. The effects of PBMT on chondrocytes have been studied both in vitro and in vivo. However, outcomes vary among reports due to the application of different light parameters.

Methods

Normal human chondrocytes growing in 96 well microplates received either blue (464nm), red (625nm), or infrared (850nm) PBMT at different fluences (1 J/cm2, 3 J/cm2, 24 J/cm2) using Dynatronics Solaris 705 Plus through the probe. A cell proliferation assay kit was used to quantify the number of chondrocytes in PBMT treated and control groups. One way ANOVA and Post Hoc Bonferroni were used to compare the differences among multiple groups using SPSS 21. p<0.05 was considered to be significantly different.

Results

We found that fluences 3 J/cm2 and 24 J/cm2 of blue PBMT significantly decreased the cell viability while 1 J/cm2 of blue PBMT did not produce significant change. Fluences of 1 J/cm2 and 24 J/cm2 of red PBMT significantly increased the cell viability but 3 J/cm2 of red PBMT did not show such an effect. Surprisingly, none of the fluences tested using infrared PBMT showed any obvious change in cell viability.

Conclusion

These findings suggest that different parameters (fluences and wavelengths) of PBMT produce different outcomes with regard to chondrocyte proliferation. These findings contribute to our understanding of the biological responses of chondrocytes when exposed to PBMT and indicate that optimal parameters of PBMT need to be determined to reach desired outcomes.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Chronic Inflammatory Exposure Changes the Phenotype of Human Skeletal Muscle Cells
Authors: McManus Christina , Richardson Chante
Published: June 2021

Study Objectives

Osteopathic Manipulative Therapy (OMT) is well documented in treating myofascial pain. OMT, when used appropriately, can reduce acute and chronic pain and improve myofascial function. Chronic pain, skeletal muscle wasting, and skeletal muscle weakness have been associated with inflammatory diseases. Skeletal muscle is a target for OMT and studies have shown that skeletal muscle weakness and wasting has been associated with inflammatory diseases. However, the molecular mechanisms driving these benefits with OMT are not well understood. Our objective is to look at how chronic inflammation, utilizing TNF‐alpha, changes the phenotype of human skeletal muscle cells (hSkM) and what affects stretching, which mimics OMT, improves these phenotypical changes. We hypothesize that chronic inflammation changes the phenotype of the myofascial tissue that can be reversed by OMT.

Methods

Human skeletal muscle cells were grown in skeletal muscle cell growth medium in a 150 mm × 25 mm dish. After reaching confluence, the hSkM cells were split and grown in five T‐75 flasks. Two of these flasks served as controls. The remaining three flasks were exposed to TNF‐alpha in varying concentrations; 0.1, 0.05, and 0.025 microgram/microliters of TNF‐alpha in distilled H2O. hSkM cells were phenotyped by looking at cell shape, cell count, and organization of fluorescent biomarkers.

Results

hSkM cells changed shape in response to chronic exposure to pro‐inflammatory factor TNF‐alpha in a dose dependent manner. Compared to control, hSkM cells chronically exposed to TNF‐alpha had cell‐cell contact and organizational changes. TNF‐alpha exposure also decreased cell count in a dose dependent manner.

Conclusions

Chronic exposure to pro‐inflammatory factor TNF‐alpha changes the hSkM cell phenotype. We predict that cell stretching will reverse these phenotypical changes.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hydrogen Peroxide Causes Iron Dysregulation in C2C12 Skeletal Muscle Cells
Authors: Anderson Jacob G. , Hans Rebekah C. , Symkins Dylan D. , Hancock Chad R.
Published: June 2021

Diabetes is becoming an increasingly prevalent issue in the United States. Diabetes and insulin resistance is associated with increased levels of circulating iron and signs of iron dysregulation. Unbound iron in cells leads to increased amounts of free radicals causing damage, including insulin resistance. Ongoing research in our lab has shown that insulin resistant rats displayed iron dysregulation in liver tissue, marked by hepatic iron accumulation, with a decreased ability to store the excess iron in ferritin storage proteins. The purpose of this study is to develop a cell model of iron dysregulation to replicate findings in previous studies in order to identify the mechanism by which this iron dysregulation occurs. Using C2C12 mouse skeletal muscle cells, we investigated the effects of hydrogen peroxide on iron regulation, and its role in the development of iron dysregulation. C2C12 cells were treated with 200 μM hydrogen peroxide for 12 hours. Following treatment with hydrogen peroxide, cells were harvested for protein and iron analysis. Protein levels of transferrin receptor (TfR) and ferritin heavy chain (FHC) were measured using western blot analysis. Total iron was measured using a total iron colorimetric assay. Hydrogen peroxide treatment decreased TfR protein levels by 24±16% (p=.03). We then developed a cellular model to reflect the stress caused by elevated circulating iron levels by treating C2C12 cells with varying concentrations (0–100 μM) of FeCl3 for 24 hours. Iron treatment resulted in an increase in total cellular iron in a dose‐dependent manner. As expected, exposure to elevated iron caused a reduction in TfR expression and an increase in FHC in a dose‐dependent manner. Using this high iron model with a 24‐hour treatment of 10 μM FeCl3, we added 200 μM hydrogen peroxide during the last 12 hours of iron treatment to impose conditions of elevated oxidative stress. The combination of iron and hydrogen peroxide resulted in an increase in TfR by 46±9% (p=.05) and a decrease in FHC by 49±13% (p=0.018), showing a complete reversal compared to hydrogen peroxide treatment alone. Total iron increased by 69±6.7% (p=0.0005). Our results show that treatment with hydrogen peroxide in the presence of iron causes an accumulation of iron and a decrease in ferritin expression, potentially leading to an increase of free iron and the production of reactive oxygen species. This represents a condition of iron dysregulation and increased oxidative stress that can contribute to insulin resistance as well as other cellular pathologies.

Support or Funding Information

Supported by BYU Mentoring Enhancement Grant

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A novel mitochondria‐targeted hydrogen sulfide delivery molecule prevents uraemia and diabetes‐induced renal cell oxidative stress
Authors: Stevens Megan , Payne Molly , Innes Elayne , Torregrossa Roberta of , Wood Mark , Stevens Maarten Koeners of , Whiteman Matthew of , Oltean Sebastian of
Published: June 2021

Chronic Kidney Disease (CKD) is a disturbingly fast growing global public health problem affecting an estimated 9–16% of the world population. We and others have confirmed that cardiovascular disease is often a state of hydrogen sulfide (H2S) deficiency, coinciding with mitochondrial dysfunction and increased oxidative stress. Mitochondria‐specific H2S‐delivery molecules such as AP39 have been shown to protect the kidney during ischaemia‐reperfusion injury and during organ transplantation. Although the primary effect of endogenous and pharmacological H2S on mammalian cells appears to be overwhelmingly mitochondrial, the pathophysiological importance of mitochondrial H2S deficiency, and the potential therapeutic benefit of H2S in CKD models of uremic stress and diabetic nephropathy have not yet been explored.

To elucidate the pathophysiology of CKD‐induced uremic stress, we first investigated the effect of indoxyl sulfate (IS), a representative uremic toxin, on the production of reactive oxygen species (‘ROS’) in multiple immortalised glomerular cell lines (glomerular endothelial cell [GEnCs], podocytes [PODs], and proximal tubule epithelial cells [PTECs]). Furthermore, we investigated the effects of mitochondria‐targeted H2S delivery molecules on IS‐induced ‘ROS’ production. Preliminary data showed that IS increased ‘ROS’ production in PODs and PTECs (100 μg/ml; P<0.05) and that the mitochondria‐targeted H2S delivery molecule AP39 (100nM) at least partially prevented this. We also investigated the effect of IS treatment on cell death, and whether AP39 was able to prevent this. IS (100ug/ml) induced significant cell death after 24 hrs treatment in both GEnCs and PODs, which was prevented by treatment with AP39 (100nM) (P<0.05). In addition to this, we found that AP39 increased the phosphorylation (Ser453) of the cytoprotective AKT in GEnCs exposed to diabetic and uremic stress. AP39 also prevented the mitochondria‐cytoplasmic translocation of the pro‐apoptotic factor cytochrome c in diabetic and uremic‐stressed GEnCs (P<0.05).

These preliminary data indicate that uremic states may deteriorate renal function via mitochondrial dependent oxidative stress, which could be prevented by therapeutic H2S delivery. Indeed, the mitochondrial ROS generation, cell death, and cytochrome c release into the cytoplasm by uremic toxins and a diabetic environment in renal cells were, at least in part, reversed by AP39. Further studies are currently underway to determine whether mitochondrial H2S deficiency could be a novel clinically applicable target for treatment in (chronic) kidney disease.

Support or Funding Information

British Heart Foundation and MRC funded research.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effects of Alamandine on the NHE3 Exchanger in in vivo Proximal Tubule of Spontaneously Hypertensive Rats.
Authors: Castelo‐Branco Regiane Cardoso , Crajoinas Renato Oliveira , Malnic Gerhard
Published: June 2021

The action of Alamandine on the NHE3 exchanger contributes to understanding the etiology of hypertension in spontaneously hypertensive rats (SHR) and their controls, Wistar‐Kyoto rats (WKY). Angiotensin‐(1–7) [Ang‐(1–7)], a heptapeptide member of the renin‐angiotensin system, acts in opposition to the vasoconstrictor, proliferative, and profibrotic actions of Angiotensin II (Ang II) in the cardiovascular system and proximal renal tubule. In normotensive Wistar rats, the dose‐dependent biphasic action of Ang‐(1–7) on NHE3 of renal proximal tubular epithelium is also in opposition of the dose‐dependent biphasic action of Ang II. The presently reported effects of Alamandine were similar to those obtained with Ang‐(1–7), including vasodilation, antifibrosis, reduction of blood pressure in spontaneously hypertensive rats and control of cardiovascular functions. This study was designed to determine the effects of Alamandine on reabsorptive bicarbonate flow (JHCO3−) in in vivo middle proximal tubules. This action was evaluated by bicarbonate reabsorption (JHCO3−) of in vivo proximal renal tubules, using microperfusion with H+ ion‐sensitive microelectrodes. By means tail‐cuff plethysmography the mean values of systolic pressure were obtained in WKY [136 ± 1 mmHg (N=15)] and SHR [181± 3 mmHg (N=9)]. In WKY rats, the mean control value of JHCO3− was 2.34 ± 0.13 nmol.cm−2.s−1 (N= 14). Alamandine [10−12 M] in luminally perfused tubules caused a significant decrease of the JHCO3− to 1,10 ± 0,07 nmol.cm−2.s−1 (28); the following perfusions also significantly decreased JHCO3−: Alamandine [10−9 M] to 1.20 ± 0.13 nmol.cm−2.s−1 (22); Alamandine [10−6 M] to 1.58 ± 0,10 nmol.cm−2.s−1 (17). In SHR rats, the mean control value of JHCO3− was 2.31 ± 0.15 nmol.cm−2.s−1 (12). Alamandine [10−12 M] in luminally perfused tubules causes a significant decrease of the JHCO3− to 1.38 ± 0.07 nmol.cm−2.s−1 (18); the following perfusions also significantly decreased JHCO3−: Alamandine [10−9 M] to 1.42 ± 0.16 nmol.cm−2.s−1 (15); Alamandine [10−6 M] to 1.58 ± 0.13 nmol.cm−2.s−1 (20). So, our data are showing that in WKY and SHR rats, the inhibitory dose‐dependent effect of Alamandine on JHCO3−, demonstrates a possible inhibitory beneficial effect of Alamandine in hypertensive rats opposite to the deleterious stimulatory effects of Ang II. It is therefore reasonable to assume that, in the intact animal, the interaction of the opposite dose‐dependent effects of Alamandine and Ang II on the Na+/H+ exchanger may represent an important physiological regulation in terms of intra‐ and extracellular volume and/or pH changes.

Support or Funding Information

FAPESP; CNPq and CAPES

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Impact of vasopressin on cell‐autonomous expression of membrane transport proteins in rat distal nephron
Authors: Smorodchenko Alina , Mutig Kerim , Bachmann Sebastian
Published: June 2021

Transcellular reabsorption of NaCl in kidney thick ascending limb (TAL) is controlled by Na,K,2Cl cotransporter (NKCC2) and involves the action of luminal (ROMK) and basolateral (Kir4.1) potassium channels, a basolateral calcium sensing receptor (CaSR); paracellular transport controlled by the claudin family of proteins. A morphological cell heterogeneity has previously been described, and mosaic expression of ROMK and Kir4.1 was reported. Here we have studied TAL EM morphology and zonal as well as cell‐autonomous heterogeneity of the transport proteins at steady state and under stimulation by vasopressin (AVP; V2R agonist dDAVP for 7 or 72 h) using AVP‐deficient Brattleboro (BB) and Long Evans (LE) control rats. NKCC2, ROMK, Kir4.1, CaSR, and Claudin (Cldn)‐10 and ‐16 signals were analyzed using immunohistochemistry, in situ hybridization, EM and Western blot (WB).

TAL morphological cell heterogeneity was obvious between kidney zones, but not at the cell‐to‐cell level within a zone. NKCC2 was continuously expressed in all TAL cells throughout, whereas ROMK vs. CaSR and Kir4.1 expression showed conspicous heterogeneity in a mutually exclusive pattern. Nearly all tight junctions (TJ) of inner stripe TAL were Cldn‐10 positive. In the outermost inner stripe and outer stripe, Cldn‐10‐ and ‐16 signals were coexpressed; ROMK‐positive cells had Cld‐10‐positive TJ, whereas ROMK‐negative cells had Cldn‐16‐positive TJ. 72h dDAVP administration significantly increased the number of ROMK‐positive cells which then coexpressed Kir4.1 signal. In parallel, Cldn‐10 expressing TJ became more numerous at the expense of Cldn‐16‐positive TJ. WB analysis showed 2.2‐fold increase for ROMK, 1.3‐fold for Kir4.1, but down‐regulation for CaSR to 60%. ISH revealed no differences in mRNA abundances. No significant changes were detected after 7h.

The present results demonstrate expressional cell heterogeneity in TAL with respect to individual cell ROMK, Kir4.1, CaSR, and Cldn‐10 and ‐16 signals and reflect an AVP‐dependent adaptive mechanism in renal solute handling in the outer medulla.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Radiofrequency Renal Denervation Decreases Fibrosis in Kidney Cortex and Medulla in Spontaneously Hypertensive Rats (SHR)
Authors: Gao Juan , Denys Ian Bart , Del Valle Luis , Kapusta Daniel R.
Published: June 2021

There are over 8.3 million people in the US that have chronic kidney disease (CKD), which ultimately can progress to end stage renal fibrosis. Despite an increasing prevalence of CKD, there are no new therapies targeting fibrosis. Our previous studies showed that removal of the renal nerves by radiofrequency renal denervation (RF‐RDN) decreases blood pressure, sympathetic activity, and renal inflammation in hypertensive SHR. Renal fibrosis is a common consequence of chronic kidney inflammation. Therefore, these studies investigated whether RF‐RDN will attenuate renal fibrosis in hypertensive SHR. 22‐week old SHR (n=7/group) received bilateral RF‐RDN or Sham‐RDN (Biosense Webster Stockert 70 generator and RF‐probe). Four weeks later, rat kidneys from all animals were harvested and paraffin sections were stained for fibrosis by Masson's trichrome staining. A pathologist, who was blinded to treatment groups, evaluated each kidney section for fibrosis. The results showed that Sham‐RDN treated SHR had extensive fibrosis demonstrated by moderate thickening of Bowman's capsule, collagen deposition in glomerulus, extensive tubulointerstitial fibrosis, and segmental glomerulosclerosis. In contrast, RF‐RDN significantly reduced each of these pathological components of fibrosis in kidney cortex and medulla. Chemokines play a critical role in mediating the infiltration of immune cells and subsequent fibrosis formation. Therefore, in other studies we measured (ELISA) expression levels of Chemokine ligand 5 (CCL5), CCL2 and CXCL16 in kidney homogenates from RF‐RDN or Sham‐RDN SHR. RF‐RDN decreased CCL5 expression in kidney of SHR compared to Sham‐RDN (78.9±1.7 vs 87.9±2.7 pg/mg protein). RF‐RDN did not alter CCL5 expression in normotensive WKY (68.0±2.2 vs 68.4±3.3 pg/mg protein). These findings suggest that RF‐RDN may offer a novel therapy for renal fibrosis and CCL5 may serve as a potential therapeutic target to treat CKD.

Support or Funding Information

NIGMS of NIH U54 GM104940 LA CaTS Roadmap Scholar to JG. NIH P30GM106392 to DRK & Pilot project to JG.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dynamic changes in sodium handling in the distal nephron during sympathetic stimulation in healthy males
Authors: Petersen Johan Casper Grove , Holstein‐Rathlou Niels‐Henrik , Sørensen Charlotte Mehlin , Petersen Lonnie Grove , Jonassen Thomas
Published: June 2021

Aim

Hypovolemia, functional or by blood loss, is associated with decreased venous return and stroke volume. Baroreceptor mediated sympathetic reflexes are activated to increase heart rate to maintain cardiac output, while additional neuro‐humeral responses supress renal sodium excretion. By application of lower body negative pressure (LBNP) to induce graded central hypovolemia, we tested the hypothesis, that low‐pressure cardio‐pulmonary baroreceptors are the main efferent renal neural pathway to sodium retention in the distal part of the nephron.

Methods

We simulated mild and/or severe hypovolemia in twenty‐four healthy males (age 23 ± 3 yrs, height 182 ± 7 cm, weight 75 ± 9 kg; mean ± SD) by application of LBNP at 0 mmHg, −15 mmHg or −30 mmHg. All subjects followed a 3‐day regimen of supplemental sodium ingestion prior to the trial and ingested 600 mg Li+ p.o. 10 hours prior to the trial. Urine samples were collected every 15 minutes during 1.5 hours of LBNP application and 1 hour of recovery. The ratio of excretion, CNa/CLi, was used as a measure for changes in sodium handling in the distal part of the nephron.

Results

LBNP of −15 mmHg and −30 mmHg caused a decrease in CNa/CLi of −3.53±0.69% (P<0.0001) and −2.79±0.49% (P<0.0001), respectively. There was no significant difference in the decrease in CNa/CLi between the two interventions. Heart rate was unchanged in the −15 mmHg group, but significantly elevated in the −30 mmHg group. Stroke volume and pulse pressure were both significantly lower in the −30 mmHg group.

Conclusion

We have shown, that the significant decrease in distal sodium clearance is similar during LBNP of both −15 and −30 mmHg even though the increased arterial baroreceptor unloading is pronouncedly increased during −30 mmHg.

Our data support an efferent sympathetic neural pathway from the low‐pressure pulmonary baroreceptors in regulation of distal renal sodium excretion, which is suppressed even at mild central hypovolemia. Moderate volume depletion including activation of arterial baroreceptors did not cause further suppression of distal renal sodium excretion.
Quantitative Cellular Anatomy of the Rat Kidney
Authors: Reece Takisha , Knepper Mark
Published: June 2021

The understanding of renal physiology at a cellular level is progressing rapidly as a result of the recent introduction of techniques for measurement of mRNA levels (RNA‐Seq) and protein levels (protein mass spectrometry) in very small samples consisting of single tubules and single cells. An important question is: How can we use single‐cell and single‐tubule data to predict results of analyses carried out at a whole kidney level, e.g. western blotting? The purpose of this work is to estimate the number of each cell type present in renal tubules of a kidney from a typical 250‐g adult rat (from proximal tubule [PT] to inner medullary collecting duct [IMCD]). For this, we used quantitative structural data gleaned from various 20th century publications. These studies identify the length of each tubule segment, the number of nephrons/collecting ducts, the number of cells per mm and the protein content per mm of each renal tubule segment. The calculations account for differing lengths of loops of Henle, junctions in the connecting tubule (CNT) and IMCD, and different populations of nephrons (short‐looped [71%] and long‐looped [29%]). The calculations reveal that the 38,000 nephrons and their collecting ducts have an aggregate length of 840 meters. The total number of cells from PT to IMCD is 207 million of which 52% are PT cells, 17% are thick ascending limb (TAL) cells, 10% are distal convoluted tubule (DCT) cells, 5% are collecting duct intercalated cells (ICs) and 10% are aquaporin‐2‐expressing cells (PCs). The calculations reveal that the total protein mass of all renal tubule epithelial cells is 119 mg or 40% of the dry weight of the kidney. In terms of protein mass, percentage of individual cell types are: PT, 72%; TAL, 10%; DCT, 7%; ICs, 2%; and PCs, 5%. The average protein masses per cell (pg/cell) are: PT, 804; medullary TAL, 402; cortical TAL, 287; DCT, 425; CNT, 288, cortical collecting duct PC, 212 and IMCD, 300. These calculations provide an illustration of how old data can take on a new significance in light of modern systems biology.

Support or Funding Information

This work was supported by the APS 2017 Frontiers Research Community Leader Fellowship to TR.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Control of intracellular chloride signaling by IRBIT‐mediated recruitment of multiple kinase and phosphatase pathways
Authors: vachel laura , Shcheynikov Nikolay , Yamazaki Osamu , Fremder Moran , Ohana Ehud , Nakazawa AI Yamazaki , Shin Dong Min , Son Aran , Yang Chin‐Rang , Knepper Mark , Muallen Shmuel
Published: June 2021

Cl− is the major anion and important osmolyte in most living cells and has a prominent role in cellular homeostasis and intracellular chloride (Cl−in) affect the activity of several transporters. IRBIT is a multifunctional protein that controls the activity of IP3 receptors and multiple Cl− and HCO3− transporters by unknown mechanism. In the case of the ubiquitous NBCe1‐B, IRBIT interacts with the NBCe1‐B N terminus autoinhibitory domain (AID), exposing two cryptic Cl‐interacting sites to confer regulation of NBCe1‐B by Cl−in. Here, we report that IRBIT do so by recruiting a panel of phosphatases and kinases that both regulate signaling by Cl−in and modulate NBCe1‐B conformation to open the transport pathway. Thus, IRBIT recruits the phosphatase PP1 and the kinase SPAK to control phosphorylation of S65 to control regulation of NBCe1‐B by Cl−in at a 32GXXXP36 site. IRBIT recruits the phosphatase calcineurin and the kinase CaMKII to control phosphorylation of S12 to control regulation of NBCe1‐B by Cl−in at a 195GXXXP199 site. IRBIT then acts on S232, S233, S235 to generate multiple conformations with either dephosphorylated S232/S233, S232/S235 or S233/S235. Each conformation has different activation by IRBIT and regulation by Cl−in. S232/S233/S235 are conserved in several members of the NBC transporters. Mutation of serine to alanine resulted in a fully activated conformation, while mutation of serine to phosphomimetic aspartate inhibited transport and eliminated IRBIT mediated activation, suggesting that S232/S233/S235 determine whether NBC transporter is in an opened or closed conformation. These findings together with structural modeling of NBCe1‐B uncover an intricate mechanism by Cl−in signals through IRBIT‐mediated recruitment of kinases and phosphatases to regulate key transporters involved in epithelial fluid and electrolyte secretion.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of Vasopressin‐Responsive Regulators of Aqp2 Gene Transcription
Authors: Jung Hyun Jun , Knepper Mark
Published: June 2021

The peptide hormone vasopressin regulates water excretion largely through control of the water channel protein, aquaporin‐2 (AQP2). Vasopressin regulates AQP2 in part by increasing Aqp2 gene transcription in collecting duct principal cells. Dysregulation of Aqp2 gene transcription has been implicated in a number of water balance disorders. However, how vasopressin regulates Aqp2 gene transcription is unclear. Here, we used next generation sequencing (NGS) techniques as well as prior published data to identify vasopressin‐responsive transcriptional regulators for Aqp2 gene transcription.

CTCF is a DNA‐binding protein that homodimerizes with upstream or downstream DNA‐bound CTCF molecules to create ‘insulated loops'. Genes in these insulated loops are regulated mainly via enhancer or repressor interactions within the same loop. From the CTCF ChIP‐Seq database in mouse ENCODE, we found that three genes (Faim2, Aqp2 and Aqp5) are within the same insulated loop (99 kb‐length) defined by two strong CTCF‐binding sites. To identify regulatory elements for Aqp2 gene transcription in mpkCCD cells, we carried out ATAC‐Seq to identify genomic regions accessible to Tn5 transposase (analogous to DNase sensitivity footprinting). We identified 12 transposase‐accessible regions within the Faim2‐Aqp2‐Aqp5 insulated loop, which mark likely locations of enhancer or repressor elements controlling Aqp2 gene transcription.

ChIP‐Seq was carried out for two candidate transcription factors, namely cyclic AMP‐responsive element‐binding protein (CREB) and CCAAT/Enhancer‐binding protein β (C/EBPβ) in mpkCCD cells. Vasopressin resulted in increased phosphorylation of CREB at Ser133, confirming a physiological response. Surprisingly, over multiple replicates, ChIP‐Seq identified no CREB‐binding sites within the Faim2‐Aqp2‐Aqp5 insulated loop, while CREB‐binding sites were found in the promoters of 29 transcription factors including AP‐1 transcription factors (Fos and Junb). The latter are recognized to play roles in regulation of Aqp2 gene transcription. In contrast, a strong C/EBPβ‐binding site was found just downstream from the Aqp2 gene within one of the transposase‐accessible regions identified by ATAC‐Seq. Vasopressin treatment increased C/EBPβ‐binding at this and other sites. Overall, the data are consistent with roles of C/EBPβ and CREB in regulation of Aqp2 gene transcription, although CREB effects are likely to be indirect through regulation of other transcription factor genes.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Kir5.1 Is Associated with NEDD4‐2 And Is Required for High K+ Intake Induced Inhibition of Kir4.1 and NCC
Authors: Lin Dao‐Hong , Wu Peng , Gao Zhong‐Xiuzi , Su Xiao‐Tong
Published: June 2021

Dietary K+ intake plays an important role in regulating NCC such that a high K+ intake (HK) decreases while a low K+ intake increases NCC. Our previous study has demonstrated that Kir4.1 plays an important role in mediating the effect of dietary K+ intake on NCC expression and activity since the deletion of Kir4.1 not only caused K+ wasting but also abolished the effect of dietary K+ intake on NCC. While Kir4.1 forms the heterotetramer with Kir5.1 and is responsible for providing K+ conductance of the basolateral membrane in the DCT, the function of Kir5.1 in the heterotetramer is not completely understood. GST‐pull‐down and immunoprecipitation have shown that Kir5.1 is associated with the Nedd4‐2 with its second WW domain on phosphothreonine‐binding motif (TPVT) of Kir5.1 at its c‐terminus (AA249–252). Ubiquitin assay and patch‐clamp experiments demonstrate that co‐expression of Nedd4‐2 decreases the expression of Kir4.1 and its currents in HEK293 cells transfected with Kir4.1/5.1 but not in the absence of Kir5.1. We now test the hypothesis that Kir5.1 is required for the effect of HK intake on the basolateral K+ channel activity of the DCT and apical NCC function. Immunostaining and western blot show that Kir4.1 expression is upregulated in Kir5.1−/− mice. Moreover, the patch‐clamp experiments demonstrate that the deletion of Kir5.1 increases the basolateral K+ conductance of the DCT and increased the negativity of DCT membrane potential (hyperpolarization). Renal clearance study has also demonstrated that thiazide‐induced natriuretic effect in Kir5.1−/− mice is significantly larger than in WT mice, indicating the upregulation of NCC in Kir5.1−/− mice. HK intake significantly decreases the basolateral K+ conductance in the DCT, depolarized the DCT membrane and inhibited NCC activity in WT mice. However, HK intake fails to decrease the basolateral K+ conductance in the DCT in Kir5.1−/− mice. Moreover, renal clearance experiments demonstrate that HK intake attenuates thiazide‐induced natriuresis only in WT mice but not in Kir5.1 knockout mice, suggesting that Kir5.1 is important in mediating HK intake‐induced inhibition of NCC. We conclude that Kir5.1 is a binding partner of Nedd4‐2 which plays a role in mediating the effect of HK intake on the basolateral Kir4.1/5.1 activity in the DCT. We speculate that HK intake may stimulate the Kir5.1/Nedd4‐2‐dependent down‐regulation of the basolateral Kir4.1 in the DCT thereby facilitating the down‐regulation of NCC.

Support or Funding Information

NIH‐DK115366

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of NHE3 in renal calcium handling
Authors: Poulsen Søren Brandt , Rieg Jessica Dominguez , Rieg Timo , Fenton Robert A.
Published: June 2021

In the renal proximal tubule (PT) and the intestine, passive paracellular Ca2+ reabsorption has been linked to active Na+ reabsorption via the Na+‐H+ exchanger type 3 (NHE3). Whole body NHE3 knockout mice show reduced intestinal Ca2+ absorption and significantly higher urinary Ca2+ excretion. To isolate the kidney‐specific role of NHE3 in this process, we examined Ca2+ handling in our recently generated kidney tubule‐specific NHE3 knockout mice [NHE3loxloxPax8Cre; Am J Physiol Renal Physiol. 2015;308(12):F1409‐20]. Twenty‐four‐hour metabolic cage experiments did not show significant differences in urinary Ca2+ excretion between genotypes on standard diet (control: 10.5 ± 1.4 μmol; NHE3loxloxPax8Cre: 11.3 ± 1.7 μmol, NS) in conjunction with normal plasma Ca2+ concentrations (control: 1.27 mM ± 0.01 mM; NHE3loxloxPax8Cre: 1.28 ± 0.01 μmol, NS). A high (4%) NaCl diet increased urinary Ca2+ excretion to a similar degree in both genotypes (control: 29.8 ± 6.3; NHE3loxloxPax8Cre: 28.9± 5.5, NS). Analysis of whole kidney samples for proteins involved in renal Ca2+ handling by RT‐qPCR indicated increased expression of transient receptor potential cation channel subfamily V member 5, TRPV5, and Na+‐Cl− cotransporter, NCC (1.34‐fold and 1.44‐fold, respectively, P < 0.05) in NHE3loxloxPax8Cre vs control mice. Similar changes were detected by immunoblotting (TRPV5: 1.57‐fold; NCC: 1.27‐fold; P < 0.05). Our results indicate intact Ca2+ homeostasis in kidney‐specific NHE3 knockout mice. We speculate that compensatory mechanisms exist; however, our results also suggest that PT Ca2+ reabsorption for long‐term Ca2+ homeostasis plays a less important role than previously assumed.

Support or Funding Information

Funding to R.A.F. is provided by the Novo Nordisk Foundation, the Lundbeck Foundation and Danish Medical Research Council. T.R. is supported by NIDDK 1R01DK110621.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Diaphragm Muscle Function Following Mid‐Cervical Contusion Injury in Rats
Authors: Khurram Obaid Ul Haq , Fogarty Matthew Joseph , Rana Sabhya , Vang Pangdra , Sieck Gary Charles , Mantilla Carlos Bernardo
Published: June 2021

Mid‐cervical spinal cord contusion injury results in tissue damage, disruption of spinal pathways, and motoneuron loss. Phrenic motoneurons located in C3–5 segments of the cervical spinal cord innervate the diaphragm muscle (DIAm), and unilateral C4 contusion results in loss of 40–50% of motoneurons ipsilateral to the injury (~25% of the total motoneuron pool). Over time after unilateral C4 spinal cord contusion injury, DIAm electromyography (EMG) increases both contralateral and ipsilateral to the side of injury, suggesting compensation due to an increased activation of the surviving motoneurons. However, whether C4 contusion impairs the ability of the DIAm to accomplish higher force motor behaviors is unknown. Transdiaphragmatic pressure (Pdi) was measured across motor behaviors over time after unilateral C4 contusion injury. Maximum Pdi (Pdimax) was elicited by bilateral phrenic nerve stimulation at 7 days post‐injury. We hypothesized that Pdimax is reduced following C4 mid‐cervical contusion injury, which will constrain the Pdi generated during different motor behaviors. Since ventilatory behaviors of the DIAm require <50% Pdimax, we further hypothesized that Pdi generated during ventilatory behaviors of the DIAm is not impaired after unilateral C4 mid‐cervical spinal cord contusion injury. In support of our hypothesis, we observed that Pdimax was reduced by ~25% after C4 mid‐cervical spinal cord contusion injury compared to a laminectomy control group. This decrease in Pdimax is consistent with the extent of phrenic motoneuron loss following contusion injury. We also found that during both eupnea (quiet breathing) and breathing stimulated by 10% O2 (hypoxia) and 5% CO2 (hypercapnia), Pdi generation was unimpaired by C4 mid‐cervical spinal cord contusion injury, again consistent with the lower force requirement of these ventilatory motor behaviors. Prior to injury, Pdi generated during airway occlusion was ~40% of Pdimax. One day following contusion injury, the Pdi amplitude during airway occlusion was reduced from ~30 cm H2O to ~20 cm, but this reduction was completely reversed by 7 days post‐injury. The reduction in Pdi amplitude at one day post‐injury cannot be attributed to the ~25% loss of phrenic motoneurons, and thus, may reflect a disruption of input to phrenic motoneurons or acute inflammation of their surrounding milieu. Over time after injury, changes in the balance between inhibition and excitation may result in recovery of higher‐force behaviors.

Support or Funding Information

Funded by NIH R01‐HL096750 and T32‐HL105355

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
OFFSPRING OF OBESE DAMS PRESENT CHANGES IN RESPIRATORY AND SYMPATHETIC ACTIVITIES
Authors: Karlen‐Amarante Marlusa , Zoccal Daniel B. , Menani José V. , Colombari Eduardo , Colombari Debora S. A.
Published: June 2021

Obesity in dams is associated with the development of several pathologies in the offspring, including cardiovascular and hormonal dysfunctions. In this study, we investigated the pattern of respiratory and sympathetic activities in the offspring of dams treated with a high‐fat diet (HFD − 45% fat). Adult female Holtzman rats (250 g) were fed with standard diet (SD) or HFD during 6 weeks before, during gestation and lactation. After weaning, male offspring of SD dams (O‐SD) or HFD dams (O‐HFD) were fed with SD. At P28–32, O‐SD and O‐HFD rats were surgically prepared to obtain the arterially‐perfused in situ preparation to examine the thoracic sympathetic, phrenic, hypoglossal and abdominal activities under basal conditions and during peripheral chemoreceptors activation (KCN −0.05%). O‐HFD rats exhibited augmented baseline phrenic inspiratory (0.80 ± 0.04 vs O‐SD: 0.57 ± 0.07 s; n=6; p < 0.05) and hypoglossal pre‐inspiratory times (0.60 ± 0.11 vs O‐SD: 0.27 ± 0.08 s, n=4; p < 0.05) compared to O‐SD. Additional expiratory bursts were observed in baseline abdominal activity of O‐HFD group, indicating the presence of active expiration (8.5 ± 2.5 vs O‐SD: 1.3 ± 0.6 events/min, n = 4 p < 0.05). Moreover, O‐HF rats showed weakened baseline respiratory‐related modulation of sympathetic activity compared to O‐HFD group. In response to stimulation of peripheral chemoreceptors, O‐HFD group showed greater abdominal reflex responses (212 ± 20 vs O‐SD: 134 ± 21 %; n = 4; p<0.05), but no significant differences were noted in the others recorded nerves comparing to O‐SD. The results indicate that male offspring of HFD‐fed dams exhibit changes in baseline respiratory pattern associated with the presence of active expiration, and blunted respiratory‐sympathetic coupling. Part of these changes persist during peripheral chemoreceptor stimulation.

Support or Funding Information

FAPESP (2016/23513‐1; 2013/17251‐6) and CNPq (425586/2016‐2).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phrenic afferent stimulation modulates cardiorespiratory output in the adult rat
Authors: Streeter Kristi , Sunshine Michael , Fuller David
Published: June 2021

Anatomical work from our laboratory indicates that small and large diameter phrenic nerve afferent fibers localize to the dorsal laminae, project throughout the ipsilateral spinal gray matter and occasionally reach the contralateral dorsal horn. In ongoing experiments, we are testing the hypothesis that activation of phrenic afferent neurons modulates contralateral phrenic motor output and arterial blood pressure in the adult rat. In anesthetized and ventilated adult Sprague‐Dawley rats, one phrenic nerve was electrically stimulated using a bipolar electrode, while respiratory bursting in the contralateral phrenic nerve and arterial blood pressure were recorded. Inspiratory triggered phrenic nerve stimulation was delivered for 20 seconds (approximately 18 breaths) at 40pps using a narrow (0.1 ms) pulse width to target activation of large diameter myelinated axons, or a wide (1.0 ms) pulse width to recruit small diameter myelinated and unmyelinated afferents. The experimental protocol consisted of eight currents (10, 25, 50, 70, 90, 120, 160, 220μA) delivered in a random order, each separated by a 5 minute recovery period (i.e. no stimulation). Each stimulus current caused a progressive increase in contralateral phrenic amplitude during the stimulation period. Phrenic amplitude remained above baseline for at least 1 minute after the cessation of stimulation. At lower currents (<50μA), the wide pulse stimulation was more effective at increasing contralateral phrenic output compared to the narrow pulse stimulation, while higher currents (>50μA) elicited a similar increase in contralateral amplitude regardless of pulse width. In addition, phrenic nerve stimulation was associated with a brief decrease, followed by an increase in mean arterial blood pressure during stimulation. In separate rats, a unilateral dorsal root rhizotomy (C3, C4, C5) was performed prior to phrenic nerve stimulation to eliminate afferent input to the spinal cord. This treatment prevented both the increase in contralateral phrenic amplitude and blood pressure at all currents and both pulse widths, indicating that the observed responses are due to activation of phrenic afferents. These preliminary results confirm that activation of phrenic afferents can modulate the activity of the contralateral phrenic motor pool, and suggest that phrenic afferents are capable of initiating phrenic motor plasticity.

Support or Funding Information

1F32NS095620‐01 (KS), T32‐ND043730 (MS), SPARC OT2 OD023854 (DDF), 1 R01 NS080180‐01A1 (DDF)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.