bluuebunny/crossref_metadata_2025 huggingface dataset:
Respiratory Function in the Mdx Mouse Model of Duchenne Muscular Dystrophy: Role of Hypoxia, Stress and Immune Factors
Authors: Burns David , Manning Jennifer , O'Malley Dervla , O'Halloran Ken
Published: June 2021

Duchenne muscular dystrophy (DMD) is a genetic disease caused by defects in the dystrophin gene. Patients have severe progressive respiratory muscle dysfunction due to progressive weakness and fibrosis culminating in hypoxaemic periods due to hypoventilation. We examined respiratory function in young adult male Mdx (dystrophin deficient) mice (C57BL/10ScSnMdx/J, n=11) and wild type (WT) controls (C57BL/10ScSn, n=10). During normoxic breathing, Mdx mice had significantly reduced breathing frequency, tidal volume and minute ventilation compared to WT. Mdx mice had a significantly increased ventilatory response to acute hypoxic challenge. We also determined that young adult Mdx mice have reduced diaphragm functional capacity compared to WT, with significant decreases in tetanic force, work and power. Interestingly, Mdx diaphragm displayed tolerance to an acute (6hr) in vivo hypoxic stress, which was not observed in WT. In separate studies, co‐treatment with a monoclonal IL‐6 receptor antibody (MR1‐61; 0.3mg/kg) and the CRFR2 agonist, Urocortin 2 (30µg/kg) both administered by 6 subcutaneous injections over 2 weeks completely rescued Mdx diaphragm muscle force loss compared to saline treatment. Mdx mice have pronounced respiratory muscle dysfunction and impaired normoxic ventilation suggestive of hypoventilation. Enhanced hypoxic chemosensitivity and respiratory muscle tolerance of hypoxic stress are suggestive of adaptation to chronic hypoxaemia in young adult life. Hypoxia, stress and immune factors are implicated in diaphragm dysfunction in the Mdx mouse model of DMD.

The MR1‐61 antibody was gifted by Chugai Pharmaceuticals, Tokyo, Japan.
Diaphragm Muscle Sarcopenia is Present in Both Male and Female Mice
Authors: Greising Sarah , Medina Juan , Stowe Jessica , Mantilla Carlos , Sieck Gary
Published: June 2021

The diaphragm muscle (DIAm) must be able to generate sufficient forces to sustain ventilatory and non‐ventilatory behaviors throughout the lifespan. Age‐related decreases in DIAm fiber cross‐sectional area and specific force, i.e., sarcopenia, may impair the ability to generate transdiaphragmatic pressures (Pdi) necessary to accomplish expulsive, non‐ventilatory behaviors essential for airway clearance. We examined the effects of aging on DIAm function in both male and female mice at 6 and 24 months of age (n=79). Maximum DIAm specific force was 30% lower in old mice (P蠄0.001) and Pdi generated during non‐ventilatory behaviors was ~40% less in old mice (P蠄0.001). No differences in either DIAm force or Pdi were found between males and females. Collectively, these results indicate that DIAm sarcopenia is present in both sexes and significantly affects the ability to generate Pdi needed for airway clearance in old age.

Supported by R01‐AG044615, T32‐HL105355, and the Mayo Clinic.
Pyk2‐Induced Tyrosine Phosphorylation of STIM1 at Y361 Residue Regulates Puncta Formation, Store‐Operated Calcium Entry and Lung Vascular Permeability
Authors: Tauseef Mohammad , Sharma Tiffany , Malik Asrar , Mehta Dolly
Published: June 2021

Store‐operated Ca2+ entry (SOCE) through transient receptor potential canonical channels (TRPCs) regulate the increase in intracellular calcium (Ca2+) in endothelial cells. Upon endoplasmic reticulum (ER) Ca2+ store depletion, STIM1 has been shown to form puncta and translocation near plasma membrane to induce SOCE. Yet, mechanisms regulating STIM1 oligomerization and translocation to the plasma membrane fundamental to activation to SOCE remain unclear. We show that thrombin as well as thapsigargin (a direct activator of SOCE) rapidly induced the tyrosine phosphorylation of STIM1 in lung endothelial cells in association with STIM1 puncta formation. STIM1 protein contains six tyrosine residues (Y98, Y130, Y150, Y231, Y316, Y361). Mutating all tyrosine residues in STIM1 protein to phenylalanine (Y‐F) suppressed SOCE. Intriguingly, mutation of Y361àF in STIM1 that lies within oligomerization domain also abrogated SOCE. We identify proline‐rich tyrosine kinase (Pyk2) as the kinase inducing STIM1 phosphorylation. We also show that overexpression of Y361‐phosphodefective mutant in WT mice endothelial cells (ECs) using liposomes conjugated with endothelial‐specific VE‐cadherin promoter cDNA prevented LPS induced pulmonary vascular permeability. Thus, our studies suggest that STIM1 phosphorylation at tyrosine (Y) 361 residue induces puncta formation and thereby play a key role in regulating SOCE and increase in endothelial permeability.
Role for Rictor/mTORC2 Signaling in Mediating TGFb1‐induced Fibroblast Activation and Kidney Fibrosis
Authors: dai chunsun
Published: June 2021

The Mammalian target of rapamycin (mTOR) is recently identified with two structurally distinct multiprotein complexes. MTORC1 has been implicated in the pathogenesis of several types of kidney disease. The role and mechanisms for mTORC2 in TGFb1‐induced fibroblast activation and kidney ﬁbrosis have not been reported. Here, we found that TGFβ1 could activate Rictor/mTORC2 signaling in cultured NRK‐49F cells, a rat kidney ﬁbroblast cell line. Blocking Rictor/mTORC2 signaling with either Rictor or Akt1 small interfering RNA transfection markedly inhibited TGFβ1‐induced fibronection and a‐SMA expression. In addition, Western blotting or immuno‐staining results showed that Rictor/mTORC2 signaling was activated in kidney interstitial myoﬁbroblasts from mice with unilateral ureter obstruction (UUO) nephropathy and from patients with various types of kidney disease including diabetic nephropathy, IgA nephropathy, lupus nephritis as well as membranous nephropathy. To further investigate the role of Rictor/mTORC2 signaling in fibroblast activation and kidney fibrosis, a mouse model with fibroblast‐specific deletion of Rictor was generated with Cre/LoxP system. The knockouts were born normal and no obvious kidney dysfunction or kidney morphologic abnormality was found within 5 months after birth. However, kidney interstitial fibrosis and inflammatory cell infiltration were markedly diminished in the knockouts at 1 and 2 weeks after UUO compared to those in their control littermates. Together, these results suggest that Rictor/mTORC2 signaling activation mediates TGFb1‐induced ﬁbroblast activation and contributes to the development of kidney ﬁbrosis, possibly providing a therapeutic target for chronic kidney diseases.
Loss of α(E)‐catenin‐Fscn2 signaling Increases Cisplatin‐Induced Apoptosis in Aged Kidney
Authors: WANG XINHUI , Nichols LaNita , Grunz‐Borgmann Elizabeth , Parrish Alan
Published: June 2021

Aging patients are highly susceptible to acute kidney injury. Previous studies in our laboratory demonstrated a dramatic decrease of α(E)‐catenin expression in proximal tubular epithelium in the aged kidney. We created stable α(E)‐catenin knock‐down NRK‐52E (C2) cells (NT3 is the non‐targeted control) and observed a significant loss of viability in C2 cells as compared with NT3 cells after cisplatin challenge. In this study, we aimed to delineate the pathway by which loss of α(E)‐catenin increases cisplatin injury. Increased caspase‐8 and ‐9 activation and cytosolic cytochrome C were observed in C2 cells after cisplatin treatment. Blocking apoptosis, using caspase‐8 or ‐9 inhibitors, completely abolishes the increased susceptibility of C2 cells. Interestingly, the expression of fascin actin bundling protein 2 (Fscn2) is decreased in α(E)‐catenin knock‐down cells. Re‐expression of Fscn2 in C2 cells attenuates the increased apoptosis following cisplatin challenge. Furthermore, our in vivo study showed a significant increase in serum creatinine, KIM‐1 and in situ apoptosis levels at 72 hr after a single dose of cisplatin in 24‐month‐old rats, but not in 4‐month‐old rats. The expression of Fscn2 was also decreased in aged kidney. Taken together, these results suggest that loss of α(E)‐catenin‐Fscn2 signaling increases cisplatin‐induced apoptosis in aged kidney.
Systemic Increase in Serum‐ and Glucocorticoid‐Inducible Kinase 1 (SGK1) Activity Potentiates Mineralocorticoid/NaCl‐Induced Renal but not Cardiac Fibrosis
Authors: Sierra‐Ramos Catalina , Hernandez Guadalberto , Salido Eduardo , Alvarez de la Rosa Diego
Published: June 2021

Mineralocorticoids stimulate renal tubular salt reabsorption, induce extracellular volume expansion, and elevate blood pressure. Mineralocorticoid excess has deleterious effects on cardio‐renal function, including the development of fibrosis. The kinase SGK1 transduces part of the effects of mineralocorticoids. In order to explore the effect of a systemic excess of SGK1 activity in the development of mineralocorticoid/NaCl‐induced cardiac and renal fibrosis, we produced mice carrying one additional genomic copy of the mouse sgk1 gene as bacterial artificial chromosome (BAC) transgene (Tg.SGK1) modified with a mutation rendering the kinase constitutively active. Tg.SGK1 mice and wild‐type littermates were uninephrectomized, treated with DOCA (75 mg/Kg) and 1% NaCl in the drinking water for 6 weeks. The treatment led to a significant increase in blood pressure slightly more pronounced in Tg.SGK1 mice. Histology revealed significantly greater glomerular, perivascular and tubulointerstitial fibrosis in Tg.SGK1 mice. In contrast, cardiac fibrosis developed without significant differences between genotypes. In the absence of treatment, Tg.SGK1 mice displayed enlarged glomeruli and increased glomerular collagen content. Our results demonstrate that increased SGK1 expression is sufficient to induce glomerular damage without added stimuli and enhances tissue specific mineralocorticoid/ NaCl induced fibrosis.
Gene Duplication and Sequence Variants of the Rattus norvegicus Y‐Chromosome can Alter Kidney Function
Authors: Prokop Jeremy , Lazar Jozef , Underwood Adam , Jacob Howard
Published: June 2021

The Y‐chromosome of mammals contributes to numerous diseases with sex disparities such as those of the cardiovascular system and renal function; however, development of model organisms to study these diseases is limited. Y‐chromosome consomics of the SHR or the SS rat strains result in altered kidney function, suggesting genetic contributions of the different rat stain Y‐chromosomes to disease development. Genetic analysis of the Y‐chromosome in whole genome sequencing of 18 male rat strains (including the SHR and SS) relative to 5 female rat strains has identified many genetic variants on the Y‐chromosome genes in addition to identifying 9 autosomal genes that have duplicated onto the rat Y‐chromosome. Analysis of RNAseq for ten tissues over four ages of the rat showed expression profiles (u=ubiquitous expression, t=testis specific expression) for EIF2S3Y (u), ZFY (t), USP9Y (t), DDX3Y (u), UTY (u), UBE1Y (t), KDM5D (u), RBMY (t),MED14Y (retroposed gene, u), UBE2Q2Y (retroposed gene, u),and Sry (eight gene copies with an additional copy in the SHR strain, u). A variant (P76T) in the Sry3 gene copy (expressed in the kidney) is able to elevate blood pressure through regulation of the renin‐angiotensin system (elevation blocked with Olmesartan) and may be a contributing factor to the SHR Y‐chromosome blood pressure elevation. One of the most interesting observations made during our analysis of the Y‐chromosome genes in the rat was that 104 week old rats have an expression profile for Y‐chromosome genes that is significantly altered from that of the 21 week old rats (in both the testis and the kidney), suggesting correlations between aging and Y‐chromosome contributions to physiology.
Aldosterone Upregulates Tight Junction Expression of Claudin‐4, Decreasing Paracellular Na+ Permeability in the Renal Cortical Collecting Duct
Authors: Rajagopal Madhumitha , Yu Alan
Published: June 2021

Under conditions of low dietary salt intake, plasma aldosterone levels are upregulated, increasing Na+ reabsorption in the renal cortical collecting duct (CCD). While aldosterone is known to enhance transcellular Na+ reabsorption, its effect on paracellular transport remains unknown. Paracellular transport is mediated by tight junction (TJ) proteins called claudins. We used a murine CCD cell line, mpKCCD c14, to study the effect of aldosterone on paracellular transport. We found that aldosterone significantly increased transepithelial resistance (TER) and decreased paracellular Na+ permeability (PNa), when transcellular transport is blocked. We also found that aldosterone increased TJ expression of a Na+ barrier claudin, claudin‐4, suggesting that increased claudin‐4 expression at the TJ is responsible for the decrease in PNa. To confirm this, we stably knocked–down claudin‐4 expression using shRNAs. This diminished both baseline TER and aldosterone‐induced increases in TER, and PCl/PNa.

Aldosterone is known to signal via a mineralocorticoid receptor and activate a serum glucocorticoid kinase (Sgk1) to enhance ENaC activity. We found that aldosterone induced changes in claudin‐4 expression and TER were also suppressed by treatment with RU486 (a steroid receptor blocker) and GSK650394 (aSgk1 antagonist). In summary, our data strongly suggests that aldosterone acts via a Sgk1 mediated pathway to upregulate expression of claudin‐4 at the TJ in the renal CCD, decreasing PNa, preventing back‐leak of actively reabsorbed Na+, and increasing net salt reabsorption. Consistent with our in‐vitro data, we have also shown that in mice fed a low salt diet renal claudin‐4 expression is enhanced.
Spinal Cord Injury Upregulates TRPM4 in the Urinary Bladder
Authors: Kullmann F Aura , Beckel Jonathan , Nakagomi Hiroshi , Wolf‐Johnston Amanda , Gauthier Christian , Kanai Anthony , Birder Lori
Published: June 2021

Spinal cord injury (SCI) profoundly alters bladder function. Chronically, it induces urothelial hyperplasia, smooth muscle hypertrophy and detrusor hyperactivity, all of which prevent the bladder from properly storing and releasing urine. Transient receptor potential melastatin 4 (TRPM4) is a non‐selective cation channel activated by intracellular Ca2+ and modulated by ATP. This channel has been implicated in various pathologies including SCI, inflammation, axonal degeneration and cardiac myocyte hypertrophy. We hypothesize that TRPM4 may play a role in bladder dysfunction after SCI. Here, we evaluated the expression and function of TRPM4 in the bladder urothelium and smooth muscle after SCI.

Bladders were removed from control and spinal cord transected C57Bl6 female mice (T8‐T9; 1‐42 days post SCI). The mucosa and smooth muscle were separated surgically and used for RT‐PCR, western blot analysis, urothelial cell culture and muscle strips contractility studies.

TRPM4 mRNA and protein were expressed in the mucosa and detrusor and were upregulated after SCI. Expression levels peaked at 3‐7 days post SCI and remained elevated for the duration of the study (6 weeks post SCI). Peak levels coincided with urothelial hyperplasia and smooth muscle hypertrophy following SCI. The TRPM4 antagonist, 9‐Phenanthrol (0.1‐30 µM), relaxed detrusor strips pre‐contracted with prostaglandin‐E2 (1µM) or carbachol (0.1µM) and reduced spontaneous activity. After SCI, the magnitude of these effects was increased.

Our data reveal that TRPM4 is expressed in the bladder mucosa and detrusor and is upregulated after SCI, supporting the hypothesis that TRPM4 is involved in the remodeling processes and/or development of detrusor overactivity following SCI.
TRPC3 Facilitates Adaptation of Collecting Duct Cells to Changes in Osmolality
Authors: Mamenko Mykola , Zaika Oleg , Boukelmoune Nabila , O'Neil Roger , Pochynyuk Oleh
Published: June 2021

Mechanical stress induced by changes in tubular flow rate and osmotic gradients is viewed to be an important regulator of the water and electrolytes transport in the collecting duct (CD). Our recent experimental effort identified that pharmacological inhibition or genetic ablation of TRPV4 precluded [Ca2+]i responses to elevated flow but not to hypotonicity suggesting involvement of discrete signaling determinants. Here we combined direct measurements of [Ca2+]i dynamics in freshly isolated split‐opened CDs with genetic tools to unravel molecular components of cellular responses to decreased osmolality. [Ca2+]i elevations elicited by hypotonicity in CD cells were abolished upon removal of extracellular Ca2+, but not by depletion of the endoplasmic reticulum stores, indicating a critical role of direct Ca2+ entry via the plasma membrane. Inhibition of Ca2+‐permeable TRPC3 channel markedly diminishes the amplitude of [Ca2+]i signal induced by hypotonic stimuli in the CD. Interestingly, genetic deletion of TRPC3 significantly increases the activation threshold for [Ca2+]i responses to changes in extracellular osmolality. Water deprivation (18 hours) greatly augmented sensitivity of CD cells to extracellular osmolality in wild‐type, but only mildly in TRPC3‐/‐ mice. At the systemic level, TRPC3 ablation results in a transient defect in urinary concentration ability in response to water deprivation. Overall, our findings demonstrate that elevated flow and osmotic gradients recruit distinct signaling cascades to increase [Ca2+]i in CD cells. Genetic ablation of TRPC3 disturbs sensitivity of CD cells to hypotonic stress contributing to delayed adaptation of the kidney to restricted water intake.
Decreased Surface NKCC2 Expression and Increased Urinary Excretion of Water and Ions in VAMP3 Knockout Mice
Authors: Caceres Paulo , Haque Mohammed , Ortiz Pablo
Published: June 2021

Trafficking of the NKCC2 cotransporter to the apical surface of thick ascending limbs (TAL) regulates excretion of urine, ions and blood pressure. We showed that vesicle fusion proteins VAMP2 and VAMP3 mediate NKCC2 exocytosis to the TAL plasma membrane. VAMP3 also mediates NKCC2 expression. We showed that VAMP3‐/‐ mice have lower blood pressure in normal and low‐Na diet. However, it is not know whether surface NKCC2 is decreased in VAMP3‐/‐ mice and the contribution of VAMP3 to renal function is unclear. We hypothesize that VAMP3‐/‐ mice have decreased surface NKCC2 expression and increased excretion of water and ions. First we studied VAMP3‐/‐ kidney histology and we found no abnormalities. Next, we measured surface NKCC2 in TAL suspensions by surface biotinylation. Surface NKCC2 was decreased by 46 ± 4% compared to wt. We also observed 23% higher urine volume (wt= 1.3 ± 0.1 ml/day vs VAMP3‐/‐ = 1.6 ± 0.2 ml/day) and lower urine osmolality (wt= 3433 ± 166 mOsm vs VAMP3‐/‐ = 2924 ± 131 mOsm). UNa, UK and UCl excretion was normal, however UCa was higher (wt= 0.60 ± 0.02 µmol/day vs VAMP3‐/‐ = 0.81 ± 0.05 µmol/day). To test whether VAMP3‐/‐ mice adjusted Na excretion after reduced Na intake, we fed them a low Na‐diet. VAMP3‐/‐ mice excreted 51% more Na than wt in 24h (wt= 39 ± 8 µmol/day vs VAMP3‐/‐= 59 ± 13 µmol/day), but at 48h they were comparable (wt= 15 ± 6 µmol/day vs VAMP3‐/‐= 20 ± 8 µmol/day). Finally, we tested whether VAMP3‐/‐ mice adjusted ion excretion after 24h of water deprivation. VAMP3‐/‐ mice properly concentrated the urine, but UNa, UCl and UK were 33%, 36% and 38% higher than wt respectively. We conclude that VAMP3 is required for surface NKCC2 expression in vivo and urine excretion in baseline conditions and during low‐Na diet or water deprivation.
Nitric Oxide Decreases the Permselectivity of the Paracellular Pathway in Thick Ascending Limbs by Elevating cGMP Levels and Activating PKG
Authors: Monzon Casandra , Garvin Jeffrey
Published: June 2021

The thick ascending limb (TAL) reabsorbs 25‐30% of the filtered load of NaCl. About 70% is reabsorbed via the transcellular pathway and 30% is reabsorbed through the Na‐selective paracellular pathway. Nitric oxide (NO) inhibits net transepithelial and transcellular Na reabsorption, but its effects on the paracellular pathway are unknown. We hypothesized that NO decreases the permselectivity of the paracellular pathway for Na in TALs via cGMP and cGMP‐dependent protein kinase (PKG). To assess relative Na/Cl permeability ratios (PNa/PCl), we perfused TALs from Sprague Dawley rats and measured dilution potentials with and without NO donors (spermine NONOate: SPM, nitroglycerin: NTG) and dibutyryl cGMP (db‐cGMP). Dilution potentials were generated by reducing the bath NaCl concentration from 141 to 32 mM and PNa/PCl was calculated using the Goldman‐Hodgkin‐Katz equation. During the control period, the dilution potential was ‐11.1 ± 2.1 mV and PNa/PCl was 2.2 ± 0.4. After SPM (200 μM) treatment, they were ‐6.5 ± 1.6 mV (n = 9; p < 0.04) and 1.5 ± 0.2 (p < 0.03), respectively. NTG produced a similar effect on both dilution potential and PNa/PCl. Time controls showed no significant changes. Db‐cGMP changed the dilution potential from ‐13.4 ± 2.9 to ‐7.5 ± 1.8 mV (n = 6; p < 0.01). In the presence of PKG inhibition with KT5823 (4 μM), SPM had no significant effect on dilution potentials (KT5823 alone: ‐8.6 ± 0.1 mV vs KT5823 + SPM: ‐7.1 ± 0.1 mV, N.S.; n = 6). KT5823 alone had no effect on dilution potentials. Phosphodiesterase 2 inhibition with BAY607550 (10 μM) did not block the effect of NO. We conclude that NO reduces the permselectivity of the paracellular pathway in TALs via cGMP and PKG. Funded by NIH HL28982.
Activation of a Novel Adaptive Mechanism Limits Renal Salt Wasting During Thiazide Inhibition of the Sodium Chloride Cotransporter (NCC)
Authors: Grimm P , Wall Susan , Delpire Eric , Welling Paul
Published: June 2021

NCC reabsorbs approximately 10% of filtered NaCl, an amount vital for maintaining extracellular electrolyte and fluid homeostasis. Thiazide inhibition of NCC is used clinically to reduce extracellular fluid volume in treatment of hypertension, but the drugs efficacy often wanes along with its natriuretic properties suggesting activation of another NaCl reabsorptive pathway. Here, we describe an adaptive process in mice given thiazides. Transcriptional profiling, together with protein abundance analysis, reveals an intercalated cell pathway synergizes with a α‐ketoglutarate (αKG) paracrine signaling system to augment sodium chloride reabsorption in distal nephron. Coordinate transcriptional activation of a multi‐gene transport system in pendrin positive intercalated cells (PPIC), involving solute carriers (Slc26a;Slc4a8; Slc4a9), carbonic anhydrase 2 & 15, and V‐type H+‐ATPase, collectively form an electroneutral NaCl reabsorption pathway. In the proximal tubule, transcript for glutamine (Slc6a19, Slc38a2, Slc38a3), glutamate (Slc7a13) and α‐KG carriers (Slc22a13, Slc13a2, Slc13a3, Slc22a6) increase, along with the enzymes (Gls & Glud1) responsible for αKG synthesis from these amino acids. Consequently, aKG in tubular fluid is elevated, as is the abundance of its GPCR (Oxgr1), which is expressed on the apical surface of PPIC, allowing paracrine delivery of αKG to stimulate the transport pathway. Identification of the integrated compensatory NaCl reabsorption mechanism provides new molecular targets for diuretics and highlights a potential cause of thiazide resistance.
The Effect of a Novel rBAT Mutation on the Expression and Function of System b0,+
Authors: Rice Sarah , Edwards Noel , Sayer John , Thwaites David
Published: June 2021

To identify the causative mutations within a cohort of cystinuria patients, the two genes (SLC3A1 and SLC7A9) encoding the subunits (rBAT and b0,+AT) of the amino acid carrier System b0,+ were analysed. A novel homozygous missense mutation (M465K) in the heavy chain chaperone protein rBAT was identified in one patient and investigation of obligate heterozygotes showed an autosomal recessive penetrance of phenotype. Homology modelling was used to create a model of the extracellular domain of rBAT based on the known crystal structure of oligo‐1,6‐glucosidase from B. cereus (PDB code 1UOK). Using this model, M465K is predicted to be located on the 7th alpha helix of the triosephosphate isomerase (TIM)‐like structure. Protein expression and function were characterised following cRNA injection in Xenopus laevis oocytes by [3H]arginine uptake, western blotting, and immunocytochemistry. Reduced protein expression and arginine uptake were observed in M465K‐injected oocytes versus wild‐type rBAT suggesting a decrease in transporter trafficking to the oocyte membrane. M465K expression was dependent upon time (post‐injection) and quantity of cRNA injected and followed a pattern similar to that observed with the neighbouring mutation M467T, the most common cystinuria‐causing mutation in several European populations. Cystinuria in this patient likely results from reduced cystine reabsorption in the renal proximal tubule due to defective trafficking of M465K and decreased System b0,+ activity in vivo. Supported by the Medical Research Council and Northern Counties Kidney Research Fund.
Activated JAK‐STAT Pathway by IL‐6 Mediates Macrophage‐induced Angiotensinogen Augmentation in Renal Proximal Tubular Cells
Authors: Satou Ryousuke , O'Leary Ryan , Miyata Kayoko , Navar L
Published: June 2021

The development of angiotensin II (Ang II)‐dependent hypertension involves increased infiltration of macrophages (MΦ) into the kidney and consequent elevation of intrarenal cytokines including interleukin 6 (IL‐6). The activated JAK‐STAT pathway has been shown to contribute to angiotensinogen (AGT) stimulation in cultured renal proximal tubular cells (PTC). Therefore, this study was performed to demonstrate that activation of JAK‐STAT pathway by IL‐6 derived from Ang II‐treated MΦ mediates AGT upregulation in PTC. Rat MΦ were treated with 0‐10‐5 M Ang II up to 48 hr. Pro‐inflammatory cytokine levels in the culture medium were determined by a multiple cytokine ELISA. IL‐6 mRNA levels were also determined by RT‐PCR. Subsequently, PTC were incubated with the collected medium from the MΦ. After the treatments, STAT3 activity and AGT expression levels in the PTC were evaluated. IL‐6 mRNA and protein expression levels were increased in Ang II‐treated MΦ (1.86 ± 0.14, protein, ratio to control). Moreover, IL‐6 levels were higher than TNF‐α and IL‐1β in the culture medium of Ang II‐treated MΦ. Elevated levels of phosphorylated STAT3 (1.78 ± 0.09, ratio to control), STAT3 nuclear trafficking, and AGT augmentation (1.69 ± 0.04, ratio to control) were observed in PTC receiving the culture medium of Ang II‐treated MΦ. Addition of a neutralizing IL‐6 antibody to the collected medium attenuated the phosphorylation of STAT3 and AGT augmentation in PTC. Additionally, AG490, a JAK2 inhibitor, also suppressed the phosphorylation of STAT3 and AGT augmentation in PTC. These results establish an important role of JAK‐STAT signaling in MΦ‐IL‐6 mediated AGT induction in the kidneys.
Expression of Transporters for Amino Acids and Glucose, GH Receptor, HSP90 and Myosin in Different Tissues of Pigs After Chronic Exposure to High Ambient Temperature
Authors: Morales Adriana , Cota Margarita , Arce Néstor , Araiza Alfonso , Cervantes Miguel
Published: June 2021

Acute response of animals exposed to artificial high ambient temperature (AT) is associated with molecular changes aimed to maintain normal cellular function and metabolism. But little information is available regarding the effect of chronic exposure (10 days or more) to natural high AT. A 21‐d study was conducted with 18 pigs (32.3 ± 0.6 kg BW) to examine the effect of natural exposure to high AT on expression of transporters for amino acids (y+L, B0AT1, and b0,+AT) and glucose (SLGT1 and GLUT4), heat shock protein 90 (HSP90), GH receptor (GHR) and myosin, in jejunum, liver, Longisimus (LM) and Semitendinosus (SM) muscles. Treatments were: 1, control pigs (room temperature, 22±2 °C) fed ad libitum (C‐AL); T2, as in T1, but feed‐restricted (C‐R); T3, pigs housed in a room with no climate control (29 to 38 °C; HS). HS and C‐R pigs consumed about 65% of that of C‐AL pigs. All pigs consumed the same diet and purified water. At the end of the study, tissue samples were collected and gene expression was analyzed by qPCR. In jejunum C‐R pigs expressed more SGLT1 (P=0.076) and y+L (P<0.001) than C‐AL pigs, but HS pigs expressed less y+L (P=0.010) and B0AT1 (P=0.012) than C‐R pigs. Expression of myosin (P=0.043) and GLUT4 (P=0.005) in LM was lower in HS than C‐R pigs, but there was no effect on SM. Chronic exposure to high AT differently affects gene expression but compared to reports regarding acute response, only a few genes are affected by chronic exposure to high AT. (Supported by CONACYT‐México).
Murine circumvallate papilla taste bud organoid development and characterization of stem/progenitor cell cycle
Authors: Aihara Eitaro , Montrose Marshall
Published: June 2021

There are limited models to study taste function, and here we generate taste bud‐like structures (“organoids”) from native circumvallate papilla tissue as well as single flow‐sorted Lgr5+ cells that develop structural and phenotypic characteristics similar to native tissue. Organoids have a multilayered epithelium with CD44+ and SOX9+ stem/progenitor in the outer layers and taste receptor‐expressing and gustducin‐expressing taste cells in the inner layers. 3D time lapse monitoring of taste bud organoids created from cell cycle reporter mice (FUCCI2 transgene G1: mVenus, S/G2/M: mCherry) or H2B‐GFP transgenic mice, shows that stem/progenitor cells migrate to form taste bud‐like structures and have cyclical 24 hrs of peaks of proliferation. During tracking of individual cells, however, a diversity of several cell cycle durations, were noted; 12.5 ± 0.3, 22.2 ± 0.7, 31.8 ± 0.6, 42.0 ± 0.9, and >50 hr, in 41.2, 23.5, 20.6, 8.8, and 2.9 percent of total analyzed cells, respectively. In organoids with cell numbers <400, 42% daughter cells had shorter cell cycle duration than their parent cells. In organoids with cell numbers ranging between 400‐800, 40% of daughter cells mimicked their parent cell cycle time. In organoids >800 cells, parental cells tended to have longer cell cycle than daughter cells. In conclusion, we resolve multiple distinct populations of replicating cells in taste bud development, and suggest that spatial and cell confluence feedback mechanisms impact progenitor cell growth. A population of rapidly cycling cells have the capacity to migrate up the taste bud niche, with implications on the mechanisms of homeostatic turnover of taste cells.
Extracellular Ubiquitin Modulates Cardiac Fibroblast Phenotype and Function
Authors: Scofield Stephanie , Daniels Christopher , Dalal Suman , Singh Mahipal , Singh Krishna
Published: June 2021

Stimulation of β‐adrenergic receptor (β‐AR) increases extracellular levels of ubiquitin (UB), and extracellular UB inhibits β‐AR‐stimulated apoptosis. In the heart, exogenous UB affects β‐AR‐stimulated increases in left ventricular function, and decreases myocardial apoptosis and fibrosis. Cardiac fibroblasts are vital for maintaining the normal function of the heart, and are integral in the structural remodeling of the heart in response to injury or stress. Here we used adult rat cardiac fibroblasts to test the hypothesis that UB modulates cardiac fibroblast phenotype and function, thereby decreasing myocardial fibrosis. Using FITC‐labeled UB and confocal microscopy, we first provide evidence that extracellular UB is internalized into cellular compartments within 30‐60 min. Immunocytochemical analysis using anti‐Ki‐67 antibodies showed that UB treatment inhibits the proliferation of actively growing fibroblasts within 24 h. UB treatment decreased the formation of lamellipodia and filopodia projections while increasing actin stress fiber formation as analyzed by FITC‐phalloidin and α‐smooth muscle actin staining. UB treatment reduced transforming growth factor β‐1 (TGF‐β1)‐stimulated migration of fibroblasts into the wound as examined by in vitro wound (scratch) assay. These results suggest a novel role of UB in modulation of cardiac fibroblast phenotype and function, and provide insight into the mechanism by which exogenous UB may modulate myocardial fibrosis.

Supported by Department of Veterans Affairs (BX002332) and NIH.
Increased Cerebrovascular Protein Transcytosis and Amyloid‐β Deposition during Hyperfibrinogenemia Alter Short‐term Memory
Authors: Muradashvili Nino , Rodriguez Walter , Maldonado Claudio , Tyagi Suresh , Lominadze David
Published: June 2021

Many inflammatory and cognitive disorders including g Alzheimer's disease are accompanied by elevated blood level of fibrinogen (Fg), called hyperfibrinogenemia (HFg). Previously we showed that HFg increases microvascular permeability mainly affecting the transcellular transport pathway. In the present study, we tested the hypothesis that HFg increases cerebrovascular protein crossing via caveolar transcytosis leading to an increased formation of Fg and amyloid‐β (Aβ) complex, which results in loss of short‐term memory.

Changes in cerebrovascular permeability were assessed in male wild type (WT, C57BL/6J) and transgenic HFg mice using a dual‐tracer probing method. In addition, in HFg mice brain vascular protein transcytosis was assessed in the presence or absence of in vivo siRNA against Caveolin‐1, which was encapsulated in liposomes that fuse with endothelium and was delivered systemically. Formation of Fg‐Aβ plaque was assessed in mouse brain cryo‐sections by immunohistochemistry. Short‐term memory of mice was estimated by a novel object recognition test (NORT).

Results indicate that increased cerebrovascular protein leakage occurs mainly through caveolar transcytosis during HFg and it is greater than what is observed in control mice. Formation of Fg‐Aβ complex was increased in HFg mice compared to that in WT animals. NORT assessment demonstrated that HFg intensifies the loss of short‐term memory in mice.

In conclusion, HFg increases cerebrovascular permeability and enhances Fg‐Aβ complex formation, which intensify short‐term memory loss and suggests a functional role of Fg in vasculo‐neuronal pathology.
H2O2 Slows GFP‐Occludin Dynamic Mobility into MDCK Cell Tight Junctions
Authors: Amsler Kurt , Janosevic Danielle , Axis Josephine
Published: June 2021

Tight junctions (TJ) located at the apicolateral border of adjacent epithelial cells form the primary barrier to the paracellular movement of water and solutes. Oxidative stress of renal epithelia causes increased permeability of the TJ. Previous studies have demonstrated that multiple TJ proteins move dynamically into and out of the TJ structure. We hypothesized that H2O­2 treatment alters the dynamic mobility of the TJ protein, occludin, leading to increased TJ permeability. In this study, we examined the effect of H2O2 treatment on the mobility of occludin protein into and out of the TJ region by Fluorescence Recovery After Photobleaching. GFP‐occludin expressed in MDCK cells was localized within the TJ region and along the lateral and apical membranes. A portion of the TJ region was photobleached and the rate of recovery of fluorescence into the photobleached region was monitored. Following photobleaching, GFP‐occludin diffused progressively into the TJ region. Kinetic parameters for GFP‐occludin (t1/2, immobile fraction) were estimated by curve fitting. The halftime for recovery from photobleaching (t1/2) in control cells was 51 + 11 seconds (mean + standard deviation, n=10). Treatment of MDCK cells with H2O2 produced a concentration‐dependent increase in t1/2 for fluorescence recovery (55 mM H2O2 – t1/2 = 78 + 11 seconds (n=16, p<0.001 to 0 mM H2O2); 110 mM H2O2 – 119 + 18 seconds (n=9, p<0.001 to both 0 mM and 55 mM H2O2­)). The immobile fraction was not affected by H2O2 treatment (0 mM – 46 + 8%, 55 mM – 37 + 14%, 110 mM – 44 + 11%, p>0.1). These results indicate that oxidative stress slows the dynamic mobility of GFP‐occludin into the TJ region. (supported by NIH grant 1R15DK091749)
The Role of TRPV1‐Expressing Neurons in the Control of Heart Rate During Dynamic Exercise in Rats
Authors: Tsuchimochi Hirotsugu , Inagaki Tadakatsu , Shirai Mikiyasu
Published: June 2021

Heart rate (HR) increases at the beginning of dynamic exercise, then remains constant or gradually increases in response to the exercise intensity. Both central command and the exercise pressor reflex (EPR) are thought to play an important role for this tachycardic response. Recently, the contribution of transient receptor potential vanilloid 1 (TRPV1) channel to the EPR has been reported (Smith SA et al, J Physiol 2010). However, the role of the TRPV1 channel on the exercise tachycardia during dynamic exercise remains largely unknown. We therefore examined the effect of systemic TRPV1 channel blockade or activation on the HR response to treadmill exercise in rats. Male Wistar rats were previously implanted with telemetry transmitters (TRM54P, Millar) to measure HR and arterial blood pressure (AP), and trained to run on a treadmill. Both HR and mean AP (MAP) responses to treadmill running (20 or 40 m/min, 60 s) were compared before and after administration of TRPV1 channel agonists or antagonists. Neither the TRPV1‐selective (SB366791; 3 mg/kg) nor the nonselective (ruthenium red; 5 mg/kg) antagonists affected the HR response to exercise. However, another TRPV1 selective antagonist 5′‐iodoresiniferatoxin (IRTX; 0.3 mg/kg) or a TRPV1 agonist capsaicin (20 mg/kg, sc under anesthesia) markedly attenuated the HR response to exercise. MAP responses to exercise were not affected by these drugs. Given the partial agonist‐like effect of IRTX, systemic desensitization of the TRPV1 channel‐expressing neurons attenuated the EPR‐mediated HR response during dynamic exercise in rats. In conclusion, TRPV1‐expressing neurons play an important role for the HR response during dynamic exercise.
The Caffeine Dose Response in Habitual Consumers Performing a 90‐s Wingate Test
Authors: Smith Noah , Ogden Kevin , Mininger Andrew , Kieffer Scott , Haak Jodie
Published: June 2021

Caffeine is the most commonly used substance to enhance athletic performance; however, the ideal dosage of caffeine to improve long anaerobic exercise is unknown. The purpose of this study was to investigate the caffeine dose response in habitual consumers using the 90‐s Wingate Anaerobic Test (WAnT90). Seven (2 F, 5 M; mean+SD: age = 20.4+1.0 y; body mass = 59.4+7.9 kg) recreationally fit habitual caffeine users (consumption > 2 servings/d) volunteered for a randomized counterbalanced double‐blind study. Subjects ingested 3, 5, or 7 mg.kg‐1 caffeine or placebo (PLB) 1h prior to testing. The testing protocol included a 3 min warm‐up and a WAnT90 with a resistance of 0.05kg.kg‐1. Peak power (PP), total power (TP), and total power decline (PD) as well as TP and PD for each 30‐s interval were calculated (TP30, TP60, TP90, PD30, PD60, and PD90). Rating of perceived exertion (RPE) was recorded following each test. One‐way ANOVA was conducted to compare each dosage of caffeine with each variable across the WAnT90. Caffeine intake log analysis revealed that the subjects were habitual users with 4.0 + 1.7 servings/day. No significant differences were found for PP and TP however, a significant increase in PD was seen for 7 mg.kg‐1 caffeine. No significant differences were found for each 30‐s interval across the test (TP30, TP60, TP90, PD30, PD60, and PD90) and there were no differences in RPE among doses. Although previous research on collegiate athlete habitual caffeine consumers showed improvement in power output for anaerobic exercise, our research on recreationally fit consumers showed 7 mg.kg‐1 to be associated with an increase in fatigue. Thus, more research is needed to confirm the effects of caffeine with long anaerobic activity and the efficacy of specific dosing strategies.
Higher‐Volume Resistance Exercise Preferentially Increases Androgen Receptor‐DNA Binding and Canonical Wnt/β‐Catenin Signaling in Resistance‐Trained Men
Authors: Spillane Micheil , Schwarz Neil , Willoughby Darryn
Published: June 2021

The purpose of the study was to determine the effect of single bouts of lower‐body, lower‐volume (LBLV) and total‐body, higher‐volume (TBHV) resistance exercise on serum testosterone (T) levels and the subsequent effects on muscle T, dihydrotestosterone (DHT), androgen receptor (AR) protein content, and AR‐DNA binding. A secondary purpose was to determine the effects on serum Wnt4 levels and skeletal muscle β‐catenin content. In a randomized cross‐over design, venous blood was obtained in resistance‐trained males immediately before and after, and after 0.5, 1, 2, 3, and 24 hr following a single bout of exercise. Muscle samples were obtained immediately before and 3 and 24 hr after exercise. Exercise bouts consisted of a LBLV and TBHV protocol, and each bout was separated by one week. Statistical analyses were performed by separate two‐way factorial analyses of variance (ANOVA) with repeated measures. No significant differences were observed in serum total and free T and skeletal muscle T and DHT with either protocol (p > 0.05). AR protein was significantly increased at 3 hr post‐exercise and conversely decreased at 24 hr post‐exercise for TBHV, whereas AR‐DNA binding was significantly increased at 3 and 24 hr post‐exercise (p < 0.05). Also in response to TBHV, serum Wnt4 was significantly increased at 0.5, 1, and 2 hr post‐exercise (p < 0.05) and β‐catenin was significantly increased at 3 and 24 hr post‐exercise (p < 0.05). It was concluded that neither volume of resistance exercise increased serum T, but that TBHV resistance exercise preferentially increased Wnt4/β‐catenin signaling and AR‐DNA binding that occurred independent of increases in serum and muscle androgen levels.
The Affective Modulation of Exercise Task Performance: Rating the Urge‐to‐Stop
Authors: Touma Loren , Martin Cameron , Smith Jordan , Kirby Briggs , Davenport Paul
Published: June 2021

Exercise performance is the integration of the physical load, the physiological response and the psychophysiological assessment of the task. Affective state modulates perceptions and motivation for the task performance. We hypothesized that the subject's rating of their Urge‐to‐Stop (RUS) in a backpack walking task will be modulated by affective responses and there will be subgroups of high responders (HiR) vs. low responders (LoR). Each subject participated in a control (no backpack weight) and experimental trial (backpack weight=40% body weight). Subjects completed a 1.6 mile exercise task on a treadmill with 8 periodic inclines of 0.1 mile. SAM scores were obtained before and after the walking task. Participants O2 consumption (VO2), CO2 production (VCO2), heart rate (HR), respiratory rate (RR) and psychophysical perception measures were recorded at the end of each incline. There were no significant perceptual differences among participants with the control task. There were significant differences in subject perceptual of RUS with experimental task. There were 2 subgroups with significant differences in RUS. The RUS increase during the loaded backpack trial in LoR was significantly less than HiR. There were no significant HiR‐LoR differences in the HR, RR, VO2 and VCO2 response to the increased load exercise tasks. SAM results for the HiR had decreased valence, increased arousal and decreased control. These results suggest that RUS during an exercise task includes an affective perceptual element and HiR greater affective responses to the task may have contributed to a higher RUS. These results also suggest the affective state of the participant is a determinant of the subject task motivation and may determine, in part, exercise performance.
NFAT5 Expression in Circulating PBMCs is a Potential Biomarker of Whole‐body Dehydration and Stress
Authors: Munoz Colleen , McKenzie Amy , Bartley Jenna , Kunces Laura , Creighton Brent , Varanoske Alyssa , Armstrong Lawrence , Maresh Carl , Lee Elaine
Published: June 2021

Background

Osmotic stress‐specific transcription factor NFAT5 (Nuclear Factor of Activated T cells) and heat stress‐specific factor HSF1 induce transcription of HSP70 and other protective proteins. Our previous work suggests NFAT5 as a novel cellular marker of whole‐body dehydration. We aimed to further characterize NFAT5, HSF1, and chaperone responses to progressive stress (dehydration, exercise, and heat) and recovery in humans.

Methods

Healthy males (n=36) completed an overnight (16h) fluid deprivation (‐2.0±0.9% body mass) followed by 2h of cycling (146±2 bpm; 38°C, 40%RH; ‐4.2±0.9% total body mass). Subjects then partially rehydrated (1h; +1.90±0.57% body mass) and returned 24h later after continued ad libitum rehydration. We measured cellular HSF1, NFAT5, and HSP70 in PBMCs (flow cytometry) and plasma eHSP70 (ELISA).

Results

PBMC NFAT5 expression increased post overnight fluid deprivation (20.96%; p=0.056) and post exercise (additional 3.35%; p=0.026), and decreased with partial rehydration (‐4.78%; p=0.072). Cellular and plasma HSP70 responded to stress and recovery with different patterns, and without detectable changes in cellular HSF1.

Conclusion

PBMC NFAT5 expression responded to dehydration. Cellular and plasma HSP70 responded to stress differently and may thus have different function. Elucidating immune cell molecular chaperone responses to stress exposure and recovery will aid in defining the effect of stress on immune cell function.
Peroxisome Proliferator‐Activated γ Coactivator‐1α (PGC‐1α) mRNA Expression Correlates Highly with mRNA Expression of Myosin Heavy Chain (MHC) Isoforms in Resistance‐Trained Men
Authors: Schwarz Neil , Spillane Micheil , Willoughby Darryn
Published: June 2021

Resistance‐trained men performed two separate bouts of resistance exercise of higher and lower intensity in cross‐over fashion. Total PGC‐1α, PGC‐1α1, MHC I, MHC IIA, and MHC IIX mRNA expression were determined by RT‐PCR from muscle samples obtained before (PRE) and 45 min, 3 hr, 24 hr, and 48 hr post‐exercise (PE). Two‐way repeated‐measures ANOVA revealed no differences in expression between exercise intensities, thus data were pooled for correlation analyses. Pearson product‐moment correlation revealed total PGC‐1α mRNA expression was significantly correlated with MHC isoforms at PRE (MHC I: r = .85, P < .01; MHC IIA: r = .88, P < .01; MHC IIX: r = .85, P < .01), 45 min PE (MHC I: r = .89, P < .01; MHC IIA: r = .86, P < .01; MHC IIX: r = .91, P < .01) , 3 hr PE (MHC I: r = .64, P < .05; MHC IIA: r = .77, P < .01; MHC IIX: r = .68, P < .05), 24 hr PE (MHC I: r = .79, P < .01; MHC IIA: r = .78, P < .01; MHC IIX: r = .73, P < .05) and 48 hr PE (MHC I: r = .83, P < .01; MHC IIA: r = .87, P < .01; MHC IIX: r = .79, P < .01). Even higher correlations were found between the PGC‐1α1 splice variant and MHC isoforms at PRE (MHC I: r = .90, P < .01; MHC IIA: r = .97, P < .01; MHC IIX: r = .96, P < .01), 45 min PE (MHC I: r = .97, P < .01; MHC IIA: r = .90, P < .01; MHC IIX: r = .97, P < .01) , 3 hr PE (MHC I: r = .74, P < .05; MHC IIA: r = .80, P < .01; MHC IIX: r = .83, P < .01), 24 hr PE (MHC I: r = .91, P < .01; MHC IIA: r = .95, P < .01; MHC IIX: r = .93, P < .01) and 48 hr PE (MHC I: r = .94, P < .01; MHC IIA: r = .99, P < .01; MHC IIX: r = .95, P < .01). Research at the proteomic level is needed determine the mechanistic nature of the relationship between mRNA expression of PGC‐1α1 and the MHC isoforms.