Markers of enterocyte damage, microbial translocation and systemic inflammation following 9 hours of heat exposure in young and older adults.

Heat stress induced damage to the gastrointestinal barrier can induce local and systemic inflammatory reactions implicated in heat-stroke. Gastrointestinal barrier damage has been shown to be greater in older relative to young adults following hyperthermia. However, comparisons between young and older adults have been limited to brief exposures (3-h), which may not reflect the duration of heat stress experienced during heat waves. We therefore evaluated markers of intestinal epithelial damage (Log transformed intestinal fatty acid binding protein, IFABPLOG), microbial translocation (soluble cluster of differentiation 14, sCD14LOG), and systemic inflammation (tumour necrosis factor alpha, TNF-αLOG; interleukin 6, IL-6LOG; C-reactive protein, CRP) in 19 young (interquartile range: 21-27 yr; 10 females) and 37 older (68-73 yr; 10 females) adults before and after 9-h of rest in 40°C (9% relative humidity). The magnitude of the increase in IFABPLOG was 0.38 log pg/mL (95% CI, 0.10, 0.65 log pg/mL) greater in the older relative to young cohort (P=0.049) after 9-h heat exposure. At baseline both IL-6LOG and CRP concentrations were higher in the older (IL-6: 2.67 (1.5) log pg/mL, CRP: 0.28 (1.5) mg/mL) relative to the young (IL-6: 1.59 log pg/mL, SD 1.2; CRP: 0.11 mg/mL, SD 1.7) group (both P<0.001). The change in IL-6 and CRP was similar between groups following 9-h heat exposure (IL-6: P=0.053; CRP: P=0.24). Neither sCD14LOG and TNF-αLOG were different between groups at baseline nor altered after 9-h heat exposure. Our data indicate that age may modify intestinal epithelial injury following 9 h of passive heat exposure.

The biphasic activity of autophagy and heat shock protein response in peripheral blood mononuclear cells following acute resistance exercise in resistance-trained males.

PURPOSE: Autophagy and heat shock protein (HSP) response are proteostatic systems involved in the acute and adaptive responses to exercise. These systems may upregulate sequentially following cellular stress including acute exercise, however, currently few data exist in humans. This study investigated the autophagic and HSP responses to acute intense lower body resistance exercise in peripheral blood mononuclear cells (PBMCs) with and without branched-chain amino acids (BCAA) supplementation. METHODS: Twenty resistance-trained males (22.3 ± 1.5 yr; 175.4 ± .7 cm; 86.4 ± 15.6 kg) performed about of intense lower body resistance exercise and markers of autophagy and HSP70 were measured immediately post- (IPE) and 2, 4, 24, 48, and 72 h post-exercise. Prior to resistance exercise, 10 subjects were randomly assigned to BCAA supplementation of 0.22 g/kg/d for 5 days pre-exercise and up to 72 h following exercise while the other 10 subjects consumed a placebo (PLCB). RESULTS: There were no difference in autophagy markers or HSP70 expression between BCAA and PLCB groups. LC3II protein expression was significantly lower 2 and 4 h post-exercise compared to pre-exercise. LC3II: I ratio was not different at any time point compared to pre-exercise. Protein expression of p62 was lower IPE, 2, and 4 h post-exercise and elevated 24 h post-exercise. HSP70 expression was elevated 48 and 72 h post-exercise. CONCLUSIONS: Autophagy and HSP70 are upregulated in PBMCs following intense resistance exercise with autophagy increasing initially post-exercise and HSP response in the latter period. Moreover, BCAA supplementation did not affect this response.

Impacts of age, type 2 diabetes, and hypertension on circulating neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 after prolonged work in the heat in men.

PURPOSE: Prolonged work in the heat increases the risk of acute kidney injury (AKI) in young men. Whether aging and age-associated chronic disease may exacerbate the risk of AKI remains unclear. METHODS: We evaluated plasma neutrophil gelatinase-associated lipocalin (NGAL) and serum kidney injury molecule-1 (KIM1) before and after 180 min of moderate-intensity work (200 W/m2) in temperate (wet-bulb globe temperature [WBGT] 16 °C) and hot (32 °C) environments in healthy young (n = 13, 22 years) and older men (n = 12, 59 years), and older men with type 2 diabetes (T2D; n = 9, 60 years) or hypertension (HTN; n = 9, 60 years). RESULTS: There were no changes in NGAL or KIM1 concentrations following prolonged work in temperate conditions in any group. Despite a similar work tolerance, the relative change in NGAL was greater in the older group when compared to the young group following exercise in the hot condition (mean difference + 82 ng/mL; p < 0.001). Baseline concentrations of KIM1 were ~ 22 pg/mL higher in the older relative to young group, increasing by ~ 10 pg/mL in each group after exercise in the heat (both p ≤ 0.03). Despite a reduced work tolerance in the heat in older men with T2D (120 ± 40 min) and HTN (108 ± 42 min), elevations in NGAL and KIM1 were similar to their healthy counterparts. CONCLUSION: Age may be associated with greater renal stress following prolonged work in the heat. The similar biomarker responses in T2D and HTN compared to healthy older men, alongside reduced exercise tolerance in the heat, suggest these individuals may exhibit greater vulnerability to heat-induced AKI if work is prolonged.

The Potential Interplay Between HIF-1α, Angiogenic, and Autophagic Signaling During Intermittent Hypoxic Exposure and Exercise.

Berkemeier QN, Deyhle MR, McCormick JJ, Escobar KA, Mermier CM. The Potential Interplay between HIF-1α, Angiogenic, and Autophagic Signaling during Intermittent Hypoxic Exposure and Exercise High Alt Med Biol. 00:000-000, 2024.-Berkemeier QN, Deyhle MR, McCormick JJ, Escobar KA, Mermier CM. The Potential Interplay between HIF-1α, Angiogenic, and Autophagic Signaling During Intermittent Hypoxic Exposure and Exercise High Alt Med Biol. 00:000-000, 2024.-Environmental hypoxia as a result of decreased barometric pressure upon ascent to high altitudes (>2,500 m) presents increased physiological demands compared with low altitudes, or normoxic environments. Competitive athletes, mountaineers, wildland firefighters, military personnel, miners, and outdoor enthusiasts commonly participate in, or are exposed to, forms of exercise or physical labor at moderate to high altitudes. However, the majority of research on intermittent hypoxic exposure is centered around hematological markers, and the skeletal muscle cellular responses to exercise in hypoxic environments remain largely unknown. Two processes that may be integral for the maintenance of cellular health in skeletal muscle include angiogenesis, or the formation of new blood vessels from preexisting vasculature and autophagy, a process that removes and recycles damaged and dysfunctional cellular material in the lysosome. The purpose of this review is to is to examine the current body of literature and highlight the potential interplay between low-oxygen-sensing pathways, angiogenesis, and autophagy during acute and prolonged intermittent hypoxic exposure in conjunction with exercise. The views expressed in this paper are those of the authors and do not reflect the official policy of the Department of Army, DOD, DOE, ORAU/ORISE or U.S. Government.

Renal upregulation of NCC counteracts empagliflozin-mediated NHE3 inhibition in normotensive but not in hypertensive male rats.

Sodium-glucose cotransporter-2 inhibitors (SGLT2i) reduce blood pressure (BP) in hypertensive patients, yet the precise molecular mechanisms remain elusive. SGLT2i inhibits proximal tubule (PT) NHE3-mediated sodium reabsorption in normotensive rodents, yet no hypotensive effect is observed under this scenario. This study examined the effect of empagliflozin (EMPA) on renal tubular sodium transport in normotensive and spontaneously hypertensive rats (SHRs). It also tested the hypothesis that EMPA-mediated PT NHE3 inhibition in normotensive rats is associated with upregulation of distal nephron apical sodium transporters. EMPA administration for 14 days reduced BP in 12-week-old SHRs but not in age-matched Wistar rats. PT NHE3 activity was inhibited by EMPA treatment in both Wistar and SHRs. In Wistar rats, EMPA increased NCC activity, mRNA expression, protein abundance, and phosphorylation levels, but not in SHRs. SHRs showed higher NKCC2 activity and abundance of cleaved ENaC α and γ subunits compared to Wistar rats, none of which were affected by EMPA. Another set of male Wistar rats was treated with EMPA, the NCC inhibitor hydrochlorothiazide (HCTZ), EMPA combined with HCTZ or vehicle for 14 days. In these rats, BP reduction was observed only with combined EMPA and HCTZ treatment, not with either drug alone. These findings suggest that NCC upregulation counteracts EMPA-mediated inhibition of PT NHE3 in male normotensive rats, maintaining their baseline BP. Moreover, the reduction of NHE3 activity without further upregulation of major apical sodium transporters beyond the PT may contribute to the BP-lowering effect of SGLT2i in experimental models and patients with hypertension.

Impaired autophagy following ex vivo cooling of simulated hypothermic temperatures in peripheral blood mononuclear cells from young and older adults.

Hypothermia is a critical consequence of extreme cold exposure that increases the risk of cold-related injury and death in humans. While the initiation of cytoprotective mechanisms including the process of autophagy and the heat shock response (HSR) is crucial to cellular survival during periods of stress, age-related decrements in these systems may underlie cold-induced cellular vulnerability in older adults. Moreover, whether potential sex-related differences in autophagic regulation influence the human cold stress response remain unknown. We evaluated the effect of age and sex on mechanisms of cytoprotection (autophagy and the HSR) and cellular stress (apoptotic signaling and the acute inflammatory response) during ex vivo hypothermic cooling. Venous blood samples from 20 healthy young (10 females; mean [SD]: 22 [2] years) and 20 healthy older (10 females; 66 [5] years) adults were either isolated immediately (baseline) for peripheral blood mononuclear cells (PBMCs) or exposed to water bath temperatures maintained at 37, 35, 33, 31, or 4 °C for 90 min before PBMC isolation. Proteins associated with autophagy, apoptosis, the HSR, and inflammation were analyzed via Western blotting. Indicators of autophagic initiation and signaling (LC3, ULK1, and beclin-2) and the HSR (HSP90 and HSP70) increased when exposed to hypothermic temperatures in young and older adults (all p ≤ 0.007). Sex-related differences were only observed with autophagic initiation (ULK1; p = 0.015). However, despite increases in autophagic initiators ULK1 and beclin-2 (all p < 0.001), this was paralleled by autophagic dysfunction (increased p62) in all groups (all p < 0.001). Further, apoptotic (cleaved-caspase-3) and inflammatory (IL-6 and TNF-α) signaling increased in all groups (all p < 0.001). We demonstrated that exposure to hypothermic conditions is associated with autophagic dysfunction, irrespective of age or sex, although there may exist innate sex-related differences in cytoprotection in response to cold exposure as evidenced through altered autophagic initiation.

Greater hyperthermia in men with type 2 diabetes does not lead to higher serum levels of cellular stress biomarkers following exercise-heat stress.

Type 2 diabetes (T2D) is associated with worsening age-related impairments in heat loss, causing higher core temperature during exercise. We evaluated whether these thermoregulatory impairments occur with altered serum protein responses to heat stress by measuring cytoprotection, inflammation, and tissue damage biomarkers in middle-aged-to-older men (50-74 years) with (n = 16) and without (n = 14) T2D following exercise in 40°C. There were no changes in irisin, klotho, HSP70, sCD14, TNF-α, and IL-6, whereas NGAL (+539 pg/mL, p = 0.002) and iFABP (+250 pg/mL, p < 0.001) increased similarly across groups. These similar response patterns occurred despite elevated core temperature in individuals with T2D, suggesting greater heat vulnerability.

Regulation of the water channel aquaporin-2 by cullin E3 ubiquitin ligases.

Aquaporin 2 (AQP2) is a vasopressin (VP)-regulated water channel in the renal collecting duct. Phosphorylation and ubiquitylation of AQP2 play an essential role in controlling the cellular abundance of AQP2 and its accumulation on the plasma membrane in response to VP. Cullin-RING ubiquitin ligases (CRLs) are multisubunit E3 ligases involved in ubiquitylation and degradation of their target proteins, eight of which are expressed in the collecting duct. Here, we used an established cell model of the collecting duct (mpkCCD14 cells) to study the role of cullins in modulating AQP2. Western blotting identified Cul-1 to Cul-5 in mpkCCD14 cells. Treatment of cells for 4 h with a pan-cullin inhibitor (MLN4924) decreased AQP2 abundance, prevented a VP-induced reduction in AQP2 Ser261 phosphorylation, and attenuated VP-induced plasma membrane accumulation of AQP2 relative to the vehicle. AQP2 ubiquitylation levels were significantly higher after MLN4924 treatment compared with controls, and they remained higher despite VP treatment. Cullin inhibition increased ERK1/2 activity, a kinase that regulates AQP2 Ser261 phosphorylation, and VP-induced reductions in ERK1/2 phosphorylation were absent during MLN4924 treatment. Furthermore, the greater Ser261 phosphorylation and reduction in AQP2 abundance during MLN4924 treatment were attenuated during ERK1/2 inhibition. MLN4924 increased intracellular calcium levels via calcium release-activated calcium channels, inhibition of which abolished MLN4924 effects on Ser261 phosphorylation and AQP2 abundance. In conclusion, CRLs play a vital role in mediating some of the effects of VP to increase AQP2 plasma membrane accumulation and AQP2 abundance. Whether modulation of cullin activity can contribute to body water homeostasis requires further studies.NEW & NOTEWORTHY Aquaporin 2 (AQP2) is essential for body water homeostasis and is regulated by the antidiuretic hormone vasopressin. The posttranslational modification ubiquitylation is a key regulator of AQP2 abundance and plasma membrane localization. Here we demonstrate that cullin-RING E3 ligases play a vital role in mediating some of the effects of vasopressin to increase AQP2 abundance and plasma membrane accumulation. The results suggest that manipulating cullin activity could be a novel strategy to alter kidney water handling.

Effect of daylong exposure to indoor overheating on autophagy and the cellular stress response in older adults.

To protect vulnerable populations during heat waves, public health agencies recommend maintaining indoor air temperature below ∼24-28 °C. While we recently demonstrated that maintaining indoor temperatures ≤26 °C mitigates the development of hyperthermia and cardiovascular strain in older adults, the cellular consequences of prolonged indoor heat stress are poorly understood. We therefore evaluated the cellular stress response in 16 adults (six females) aged 66-78 years during 8 h rest in ambient conditions simulating homes maintained at 22 °C (control) and 26 °C (indoor temperature upper limit proposed by health agencies), as well as non-air-conditioned domiciles during hot weather and heat waves (31 and 36 °C, respectively; all 45% relative humidity). Western blot analysis was used to assess changes in proteins associated with the cellular stress response (autophagy, apoptosis, acute inflammation, and heat shock proteins) in peripheral blood mononuclear cells harvested prior to and following exposure. Following 8 h exposure, no cellular stress response-related proteins differed significantly between the 26 and 22 °C conditions (all, P ≥ 0.056). By contrast, autophagy-related proteins were elevated following exposure to 31 °C (p62: 1.5-fold; P = 0.003) and 36 °C (LC3-II, LC3-II/I, p62; all ≥2.0-fold; P ≤ 0.002) compared to 22 °C. These responses were accompanied by elevations in apoptotic signaling in the 31 and 36 °C conditions (cleaved-caspase-3: 1.8-fold and 3.7-fold, respectively; P ≤ 0.002). Furthermore, HSP90 was significantly reduced in the 36 °C compared to 22 °C condition (0.7-fold; P = 0.014). Our findings show that older adults experience considerable cellular stress during prolonged exposure to elevated ambient temperatures and support recommendations to maintain indoor temperatures ≤26 °C to prevent physiological strain in heat-vulnerable persons.

Enriched Single-Nucleus RNA-Sequencing Reveals Unique Attributes of Distal Convoluted Tubule Cells.

SIGNIFICANCE STATEMENT: High-resolution single-nucleus RNA-sequencing data indicate a clear separation between primary sites of calcium and magnesium handling within distal convoluted tubule (DCT). Both DCT1 and DCT2 express Slc12a3, but these subsegments serve distinctive functions, with more abundant magnesium-handling genes along DCT1 and more calcium-handling genes along DCT2. The data also provide insight into the plasticity of the distal nephron-collecting duct junction, formed from cells of separate embryonic origins. By focusing/changing gradients of gene expression, the DCT can morph into different physiological cell states on demand. BACKGROUND: The distal convoluted tubule (DCT) comprises two subsegments, DCT1 and DCT2, with different functional and molecular characteristics. The functional and molecular distinction between these segments, however, has been controversial. METHODS: To understand the heterogeneity within the DCT population with better clarity, we enriched for DCT nuclei by using a mouse line combining "Isolation of Nuclei Tagged in specific Cell Types" and sodium chloride cotransporter-driven inducible Cre recombinase. We sorted the fluorescently labeled DCT nuclei using Fluorescence-Activated Nucleus Sorting and performed single-nucleus transcriptomics. RESULTS: Among 25,183 DCT cells, 75% were from DCT1 and 25% were from DCT2. In addition, there was a small population (<1%) enriched in proliferation-related genes, such as Top2a , Cenpp , and Mki67 . Although both DCT1 and DCT2 expressed sodium chloride cotransporter, magnesium transport genes were predominantly expressed along DCT1, whereas calcium, electrogenic sodium, and potassium transport genes were more abundant along DCT2. The transition between these two segments was gradual, with a transitional zone in which DCT1 and DCT2 cells were interspersed. The expression of the homeobox genes by DCT cells suggests that they develop along different trajectories. CONCLUSIONS: Transcriptomic analysis of an enriched rare cell population using a genetically targeted approach clarifies the function and classification of distal cells. The DCT segment is short, can be separated into two subsegments that serve distinct functions, and is speculated to derive from different origins during development.

Changes in surrogate markers of intestinal epithelial injury and microbial translocation in young and older men during prolonged occupational heat stress in temperate and hot conditions.

PURPOSE: Exertional heat stress can cause damage to the intestinal epithelium and disrupt gastrointestinal barrier integrity, leading to microbial translocation (MT) linked to the development of heat stroke. This study aimed to assess age-related differences in markers of intestinal epithelial injury and MT following non-heat stress and high-heat stress exercise in healthy young and older men. METHODS: Markers of intestinal epithelial injury (intestinal fatty acid-binding protein-'IFABP') and MT (soluble cluster of differentiation 14-'sCD14'; and lipopolysaccharide-binding protein-'LBP') were assessed in healthy young (18-30 y, n = 13) and older (50-70 y) men (n = 12). Blood samples were collected before, after 180 min of moderate-intensity (metabolic rate: 200 W/m2) walking and following 60 min recovery in either a non-heat stress [temperate: 21.9 °C, 35% relative humidity (RH)] or high-heat stress (hot: 41.4 °C, 35% RH) environment. RESULTS: There were no differences in IFABP and sCD14 between the young and older groups in the temperate condition, while LBP was greater in the older group (+ 0.66 ug/mL; + 0.08 to + 1.24 ug/mL). In the hot condition, the older group experienced greater increases in IFABP compared to the young group (+ 712 pg/mL/hr; + 269 to + 1154 pg/mL/hr). However, there were no clear between-group differences for sCD14 (+ 0.24 ug/mL/hr, - 0.22 to + 0.70 ug/mL/hr) or LBP (+ 0.86 ug/mL/hr, - 0.73 to + 2.46 ug/mL/hr). CONCLUSION: While older men may experience greater intestinal epithelial injury following exercise in the heat; this did not lead to a greater magnitude of microbial translocation relative to their younger counterparts.

Role of Angiotensin II Type 1a Receptor (AT1aR) of Renal Tubules in Regulating Inwardly Rectifying Potassium Channels 4.2 (Kir4.2), Kir4.1, and Epithelial Na+ Channel (ENaC).

BACKGROUND: Kir4.2 and Kir4.1 play a role in regulating membrane transport in the proximal tubule (PT) and in the distal-convoluted-tubule (DCT), respectively. METHODS: We generated kidney-tubule-specific-AT1aR-knockout (Ks-AT1aR-KO) mice to examine whether renal AT1aR regulates Kir4.2 and Kir4.1. RESULTS: Ks-AT1aR-KO mice had a lower systolic blood pressure than Agtr1aflox/flox (control) mice. Ks-AT1aR-KO mice had a lower expression of NHE3 (Na+/H+-exchanger 3) and Kir4.2, a major Kir-channel in PT, than Agtr1aflox/flox mice. Whole-cell recording also demonstrated that the membrane potential in PT of Ks-AT1aR-KO mice was lesser negative than Agtr1aflox/flox mice. The expression of Kir4.1 and Kir5.1, Kir4.1/Kir5.1-mediated K+ currents of DCT and DCT membrane potential in Ks-AT1aR-KO mice, were similar to Agtr1aflox/flox mice. However, angiotensin II perfusion for 7 days hyperpolarized the membrane potential in PT and DCT of the control mice but not in Ks-AT1aR-KO mice, while angiotensin II perfusion did not change the expression of Kir4.1, Kir4.2, and Kir5.1. Deletion of AT1aR did not significantly affect the expression of αENaC (epithelial Na+ channel) and ßENaC but increased cleaved γENaC expression. Patch-clamp experiments demonstrated that deletion of AT1aR increased amiloride-sensitive Na+-currents in the cortical-collecting duct but not in late-DCT. However, tertiapin-Q sensitive renal outer medullary potassium channel currents were similar in both genotypes. CONCLUSIONS: AT1aR determines the baseline membrane potential of PT by controlling Kir4.2 expression/activity but AT1aR is not required for determining the baseline membrane potential of the DCT and Kir4.1/Kir5.1 activity/expression. However, AT1aR is required for angiotensin II-induced hyperpolarization of basolateral membrane of PT and DCT. Deletion of AT1aR had no effect on baseline renal outer medullary potassium channel activity but increased ENaC activity in the CCD.

Dose-dependent nonthermal modulation of whole body heat exchange during dynamic exercise in humans.

To maintain heat balance during exercise, humans rely on skin blood flow and sweating to facilitate whole body dry and evaporative heat exchange. These responses are modulated by the rise in body temperature (thermal factors), as well as several nonthermal factors implicated in the cardiovascular response to exercise (i.e., central command, mechanoreceptors, and metaboreceptors). However, the way these nonthermal factors interact with thermal factors to maintain heat balance remains poorly understood. We therefore used direct calorimetry to quantify the effects of dose-dependent increases in the activation of these nonthermal stimuli on whole body dry and evaporative heat exchange during dynamic exercise. In a randomized crossover design, eight participants performed 45-min cycling at a fixed metabolic heat production (200 W/m2) in warm, dry conditions (30°C, 20% relative humidity) on four separate occasions, differing only in the level of lower-limb compression applied via bilateral thigh cuffs pressurized to 0, 30, 60, or 90 mmHg. This model provoked increments in nonthermal activation while ensuring the heat loss required to balance heat production was matched across trials. At end-exercise, dry heat loss was 2 W/m2 [1, 3] lower per 30-mmHg pressure increment (P = 0.006), whereas evaporative heat loss was elevated 5 W/m2 [3, 7] with each pressure increment (P < 0.001). Body heat storage and esophageal temperature did not differ across conditions (both P ≥ 0.600). Our findings indicate that the nonthermal factors engaged during exercise exert dose-dependent, opposing effects on whole body dry and evaporative heat exchange, which do not significantly alter heat balance.NEW & NOTEWORTHY To maintain heat balance during exercise, humans rely on skin blood flow and sweating to facilitate dry and evaporative heat exchange. These responses are modulated by body temperatures (thermal factors) and several nonthermal factors (e.g., central command, metaboreceptors), although the way thermal and nonthermal factors interact to regulate body temperature is poorly understood. We demonstrate that nonthermal factors exert dose-dependent, opposing effects on dry and evaporative heat loss, without altering heat storage during dynamic exercise.

Dysregulation of the WNK4-SPAK/OSR1 pathway has a minor effect on baseline NKCC2 phosphorylation.

The with-no-lysine kinase 4 (WNK4)-sterile 20/SPS-1-related proline/alanine-rich kinase (SPAK)/oxidative stress-responsive kinase 1 (OSR1) pathway mediates activating phosphorylation of the furosemide-sensitive Na+-K+-2Cl- cotransporter (NKCC2) and the thiazide-sensitive NaCl cotransporter (NCC). The commonly used pT96/pT101-pNKCC2 antibody cross-reacts with pT53-NCC in mice on the C57BL/6 background due to a five amino acid deletion. We generated a new C57BL/6-specific pNKCC2 antibody (anti-pT96-NKCC2) and tested the hypothesis that the WNK4-SPAK/OSR1 pathway strongly regulates the phosphorylation of NCC but not NKCC2. In C57BL/6 mice, anti-pT96-NKCC2 detected pNKCC2 and did not cross-react with NCC. Abundances of pT96-NKCC2 and pT53-NCC were evaluated in Wnk4-/-, Osr1-/-, Spak-/-, and Osr1-/-/Spak-/- mice and in several models of the disease familial hyperkalemic hypertension (FHHt) in which the CUL3-KLHL3 ubiquitin ligase complex that promotes WNK4 degradation is dysregulated (Cul3+/-/Δ9, Klhl3-/-, and Klhl3R528H/R528H). All mice were on the C57BL/6 background. In Wnk4-/- mice, pT53-NCC was almost absent but pT96-NKCC2 was only slightly lower. pT53-NCC was almost absent in Spak-/- and Osr1-/-/Spak-/- mice, but pT96-NKCC2 abundance did not differ from controls. pT96-NKCC2/total NKCC2 was slightly lower in Osr1-/- and Osr1-/-/Spak-/- mice. WNK4 expression colocalized not only with NCC but also with NKCC2 in Klhl3-/- mice, but pT96-NKCC2 abundance was unchanged. Consistent with this, furosemide-induced urinary Na+ excretion following thiazide treatment was similar between Klhl3-/- and controls. pT96-NKCC2 abundance was also unchanged in the other FHHt mouse models. Our data show that disruption of the WNK4-SPAK/OSR1 pathway only mildly affects NKCC2 phosphorylation, suggesting a role for other kinases in NKCC2 activation. In FHHt models NKCC2 phosphorylation is unchanged despite higher WNK4 abundance, explaining the thiazide sensitivity of FHHt.NEW & NOTEWORTHY The renal cation cotransporters NCC and NKCC2 are activated following phosphorylation mediated by the WNK4-SPAK/OSR1 pathway. While disruption of this pathway strongly affects NCC activity, effects on NKCC2 activity are unclear since the commonly used phospho-NKCC2 antibody was recently reported to cross-react with phospho-NCC in mice on the C57BL/6 background. Using a new phospho-NKCC2 antibody specific for C57BL/6, we show that inhibition or activation of the WNK4-SPAK/OSR1 pathway in mice only mildly affects NKCC2 phosphorylation.

Body Core Temperature After Foot Immersion and Neck Cooling in Older Adults Exposed to Extreme Heat.

This randomized study evaluates the hypothesis that foot immersion in cool water alone or with supplemental neck cooling mitigates increases in core temperature in older adults exposed to environmental conditions simulating deadly heat waves in North America.

Temperature-Dependent Relationship of Autophagy and Apoptotic Signaling During Cold-Water Immersion in Young and Older Males.

Autophagy is a crucial cytoprotective mechanism preventing the accumulation of cellular damage, especially during external stimuli such as cold exposure. Older adults poorly tolerate cold exposure and age-related impairments in autophagy may contribute to the associated reductions in cold tolerance. The purpose of this investigation is to evaluate the effect of different intensities of in vivo cold-water immersion and in vitro cold exposure on autophagic and apoptotic signaling in young and older males. Peripheral blood mononuclear cells (PBMCs) are isolated at baseline, end-cold exposure, and after 3 h of thermoneutral recovery. Additionally, PBMCs are treated with rapamycin and bafilomycin prior to in vitro cold exposure equivalent to in vivo core temperatures (35-37 °C). Proteins associated with autophagy, apoptosis, the heat shock response, and inflammation are analyzed via Western blotting. Moderate cold stress (0.5 °C decrease in core temperature) increased autophagic and heat shock protein activity while high cold stress (1.0 °C decrease in core temperature) augmented apoptosis in young males. In older males, minimal autophagic activation during both cold-water exposures are associated with increased apoptotic and inflammatory proteins. Although in vitro cold exposure confirmed age-related dysfunction in autophagy, rapamycin-induced stimulation of autophagic proteins underlie the potential to reverse age-related vulnerability to cold exposure.

Physiological responses to 9 hours of heat exposure in young and older adults. Part III: Association with self-reported symptoms and mood state.

Older adults are at greater risk of heat-related morbidity and mortality during heat waves, which is commonly linked to impaired thermoregulation. However, little is known about the influence of increasing age on the relation between thermal strain and perceptual responses during daylong heat exposure. We evaluated thermal and perceptual responses in 20 young (19-31 yr) and 39 older adults (20 with hypertension and/or type 2 diabetes; 61-78 yr) resting in the heat for 9 h (heat index: 37°C). Body core and mean skin temperature areas under the curve (AUC, hours 0-9) were assessed as indicators of cumulative thermal strain. Self-reported symptoms (68-item environmental symptoms questionnaire) and mood disturbance (40-item profile of mood states questionnaire) were assessed at end-heating (adjusted for prescores). Body core temperature AUC was 2.4°C·h [1.0, 3.7] higher in older relative to young adults (P < 0.001), whereas mean skin temperature AUC was not different (-0.5°C·h [-4.1, 3.2] P = 0.799). At end-heating, self-reported symptoms were not different between age groups (0.99-fold [0.80, 1.23], P = 0.923), with or without adjustment for body core or mean skin temperature AUC (both P ≥ 0.824). Mood disturbance was 0.93-fold [0.88, 0.99] lower in older, relative to young adults (P = 0.031). Older adults with and without chronic health conditions experienced similar thermal strain, yet those with these conditions reported lower symptom scores and mood disturbance compared with young adults and their age-matched counterparts (all P ≤ 0.026). Although older adults experienced heightened thermal strain during the 9-h heat exposure, they did not experience greater self-reported symptoms or mood disturbance relative to young adults.NEW & NOTEWORTHY Despite experiencing greater cumulative thermal strain during 9 h of passive heat exposure, older adults reported similar heat-related symptoms and lower mood disturbance than young adults. Furthermore, self-reported symptoms and mood disturbance were lower in older adults with common age-associated health conditions than young adults and healthy age-matched counterparts. Perceptual responses to heat in older adults can underestimate their level of thermal strain compared with young adults, which may contribute to their increased heat vulnerability.

The intensity-dependent effects of exercise and superimposing environmental heat stress on autophagy in peripheral blood mononuclear cells from older men.

Autophagy is a vital cellular process, essential to maintaining cellular function during acute physiological stressors including exercise and heat stress. We previously showed that autophagy occurs during exercise in an intensity-dependent manner in peripheral blood mononuclear cells (PBMCs) from young men, with elevated responses in the heat. However, given autophagy declines with age, it is unclear whether a similar pattern of response occurs in older adults. Therefore, we evaluated autophagy and the cellular stress response [i.e., apoptosis, inflammation, and the heat shock response (HSR)] in PBMCs from 10 healthy older men [mean (SD): aged 70 yr (5)] in response to 30 min of semirecumbent cycling at low, moderate, and vigorous intensities [40, 55, and 70% maximal oxygen consumption (V̇o2max), respectively] in a temperate (25°C) environment, with an additional vigorous-intensity bout (70% of V̇o2max) performed in a hot environment (40°C). Responses were evaluated before and after exercise, as well as throughout a 6-h seated recovery period performed in the same environmental conditions as the respective exercise bout. Proteins were assessed via Western blot. Although we observed elevations in mean body temperature with each increase in exercise intensity, autophagy was only stimulated during vigorous-intensity exercise, where we observed elevations in LC3-II (P < 0.05). However, when the same exercise was performed in the heat, the LC3-II response was attenuated, which was accompanied by significant p62 accumulation (P < 0.05). Altogether, our findings demonstrate that older adults exhibit autophagic impairments when the same vigorous-intensity exercise is performed in hot environments, potentially underlying heat-induced cellular vulnerability in older men.NEW & NOTEWORTHY We demonstrate that autophagic stimulation occurs in response to short-duration (30-min) vigorous-intensity exercise in peripheral blood mononuclear cells from older adults; however, no changes in autophagy occur during low- or moderate-intensity exercise. Moreover, older adults exhibit autophagic impairments when the same vigorous-intensity exercise is performed in hot ambient conditions. When paired with an attenuated heat shock response, as well as elevated apoptotic responses, older men may exhibit greater cellular vulnerability to exertional heat stress.

Engineering Proteins Using Statistical Models of Coevolutionary Sequence Information.

Homologous protein sequences are wonderfully diverse, indicating many possible evolutionary "solutions" to the encoding of function. Consequently, one can construct statistical models of protein sequence by analyzing amino acid frequency across a large multiple sequence alignment. A central premise is that covariance between amino acid positions reflects coevolution due to a shared functional or biophysical constraint. In this review, we describe the implementation and discuss the advantages, limitations, and recent progress on two coevolution-based modeling approaches: (1) Potts models of protein sequence (direct coupling analysis [DCA]-like), and (2) the statistical coupling analysis (SCA). Each approach detects interesting features of protein sequence and structure-the former emphasizes local physical contacts throughout the structure, while the latter identifies larger evolutionarily coupled networks of residues. Recent advances in large-scale gene synthesis and high-throughput functional selection now motivate additional work to benchmark model performance across quantitative function prediction and de novo design tasks.

Elevations in serum brain-derived neurotrophic factor following occupational heat stress are not influenced by age or common chronic disease.

With global warming, workers are increasingly exposed to strenuous occupations in hot environments. Given age- and disease-associated declines in thermoregulatory function, older workers are at an elevated risk of developing heat-related injuries. Brain-derived neurotrophic factor (BDNF) is thought to confer neuroprotection during acute exercise, however, the influence of environmental heat on BDNF responses during prolonged work remains unclear. Therefore, we evaluated serum BDNF concentrations before and after 180 min of moderate-intensity treadmill walking (200 W/m2) and after 60 min of post-exercise recovery in temperate (wet-bulb globe temperature (WBGT) 16°C) and hot (WBGT 32°C) environments in 13 healthy young men (mean [SD; 22 [3] years), 12 healthy older men (59 [4] years), 10 men with hypertension (HTN) (60 [4] years), and 9 men with type 2 diabetes (T2D) (60 [5] years). In the temperate condition, all but one participant (1 HTN) completed the 180 min of exercise. While exercise tolerance in the heat was lower in older men with HTN (117 min [45]) and T2D (123 min [42]) compared to healthy older men (159 min [31]) (both p ≤ 0.049), similar end-exercise rectal temperatures (38.9°C [0.4]) were observed across groups, paralleled by similar elevations in serum BDNF across groups at end-exercise (+1106 pg/mL [203]) and end-recovery (+938 pg/mL [146]; all p ≤ 0.01) in the heat. No changes in serum BDNF were observed in the temperate condition. Our findings indicate similar BDNF responses in individuals with HTN or T2D compared to their healthy counterparts, despite exhibiting reduced tolerance to heat.