Brain-derived neurotrophic factor in older adults exposed to simulated indoor overheating.

PURPOSE: Brain-derived neurotrophic factor (BDNF) is a neuroprotective growth factor that increases in young adults during short, intense bouts of passive heat stress. However, this may not reflect the response in heat-vulnerable populations exposed to air temperatures more consistent with indoor overheating during hot weather and heatwaves, especially as the BDNF response to acute stressors may diminish with increasing age. We therefore evaluated the ambient and body temperature-dependent responses of BDNF in older adults during daylong passive heating. METHODS: Sixteen older adults (6 females; aged 66-78 years) completed 8-h exposure to four randomized ambient conditions simulating those experienced indoors during hot weather and heatwaves in continental climates: 22 °C (air-conditioning; control), 26 °C (health-agency-recommended indoor temperature limit), 31 °C, and 36 °C (non-airconditioned home); all 45% relative humidity. To further investigate upstream mechanisms of BDNF regulation during thermal strain, we also explored associations between BDNF and circulating heat shock protein 70 (HSP70; taken as an indicator of the heat shock response). RESULTS: Circulating BDNF was elevated by ~ 28% (1139 [95%CI: 166, 2112] pg/mL) at end-exposure in the 36 °C compared to the 22 °C control condition (P = 0.026; 26 °C-and 31 °C-22 °C differences: P ≥ 0.090), increasing 90 [22, 158] pg/mL per 1 °C rise in ambient temperature (linear trend: P = 0.011). BDNF was also positively correlated with mean body temperatures (P = 0.013), which increased 0.12 [0.10, 0.13]°C per 1 °C rise in ambient temperature (P < 0.001). By contrast, serum HSP70 did not change across conditions (P ≥ 0.156), nor was it associated with BDNF (P = 0.376). CONCLUSION: Our findings demonstrate a progressive increase in circulating BDNF during indoor overheating in older adults.

Effect of Electric Fans on Body Core Temperature in Older Adults Exposed to Extreme Indoor Heat.

This study assessed whether electric fans limit core temperature increases in adults aged 65 to 85 years exposed to conditions similar to those experienced in homes during heat waves in North America.

Computationally accelerated identification of P-glycoprotein inhibitors.

Overexpression of the polyspecific efflux transporter, P-glycoprotein (P-gp, MDR1, ABCB1 ), is a major mechanism by which cancer cells acquire multidrug resistance (MDR), the resistance to diverse chemotherapeutic drugs. Inhibiting drug transport by P-gp can resensitize cancer cells to chemotherapy, but there are no P-gp inhibitors available to patients. Clinically unsuccessful P-gp inhibitors tend to bind at the pump's transmembrane drug binding domains and are often P-gp transport substrates, resulting in lowered intracellular concentration of the drug and altered pharmacokinetics. In prior work, we used computationally accelerated drug discovery to identify novel P-gp inhibitors that target the pump's cytoplasmic nucleotide binding domains. Our first-draft study provided conclusive evidence that the nucleotide binding domains of P-gp are viable targets for drug discovery. Here we develop an enhanced, computationally accelerated drug discovery pipeline that expands upon our prior work by iteratively screening compounds against multiple conformations of P-gp with molecular docking. Targeted molecular dynamics simulations with our homology model of human P-gp were used to generate docking receptors in conformations mimicking a putative drug transport cycle. We offset the increased computational complexity using custom Tanimoto chemical datasets, which maximize the chemical diversity of ligands screened by docking. Using our expanded, virtual-assisted pipeline, we identified nine novel P-gp inhibitors that reverse MDR in two types of P-gp overexpressing human cancer cell lines, reflecting a 13.4% hit rate. Of these inhibitors, all were non-toxic to non-cancerous human cells, and six were not likely to be transport substrates of P-gp. Our novel P-gp inhibitors are chemically diverse and are good candidates for lead optimization. Our results demonstrate that the nucleotide binding domains of P-gp are an underappreciated target in the effort to reverse P-gp-mediated multidrug resistance in cancer.

Cullin 3/with No Lysine [K] Kinase/Ste20/SPS-Related Proline Alanine Rich Kinase Signaling: Impact on NaCl Cotransporter Activity in BP Regulation.

The sodium chloride cotransporter (NCC) fine-tunes Na + balance and indirectly affects the homeostasis of other ions including K + , Mg 2+ , and Ca 2+ . Owing to its effects on Na + balance, BP is significantly affected by alterations in NCC activity. Several factors have been reported to influence the expression and activity of NCC. One critical factor is NCC phosphorylation/dephosphorylation that occurs at key serine-threonine amino acid residues of the protein. Phosphorylation, which results in increased NCC activity, is mediated by the with no lysine [K] (WNK)-SPS-related proline alanine rich kinase (SPAK)/OSR1 kinases. NCC activation stimulates reabsorption of Na + , increasing extracellular fluid volume and hence BP. On the other hand, proteasomal degradation of WNK kinases after ubiquitination by the Cullin 3-Kelch-like 3 E3 ubiquitin ligase complex and dephosphorylation pathways oppose WNK-SPAK/OSR1-mediated NCC activation. Components of the Cullin 3/Kelch-like 3-WNK-SPAK/OSR1 regulatory pathway may be targets for novel antihypertensive drugs. In this review, we outline the impact of these regulators on the activity of NCC and the consequent effect on BP.

The effect of foot immersion and neck cooling on cardiac autonomic function in older adults exposed to indoor overheating: a randomized crossover trial.

Foot immersion and neck cooling are recommended cooling strategies for protecting heat-vulnerable persons during heat waves. While we recently showed that these strategies do not limit core temperature increases in older adults during prolonged heat exposure, we did observe small reductions in heart rate. Expanding on these findings, we examined the effects of foot immersion with and without neck cooling on cardiac autonomic function. Seventeen adults (9 females; 65-81 years) underwent 3 randomized, 6 h exposures to 38 °C and 35% relative humidity with: no cooling (control), foot immersion (20 °C water), or foot immersion with a wet towel (20 °C) around the neck. Cardiac autonomic responses were measured at baseline and end-exposure. These included heart rate variability, cardiac and systolic blood pressure responses to standing, indexed via the 30:15 ratio and supine-to-standing systolic pressure change, respectively, and baroreflex sensitivity during repeated sit-to-stand maneuvers. The 30:15 ratio was 0.04 [95% CI: 0.01, 0.07] greater with foot immersion and neck cooling (1.08 (SD: 0.04)) relative to control (1.04 (0.06); P = 0.018). Similarly, standing systolic blood pressure was elevated 9 [0, 17] mmHg with foot immersion and neck cooling (P = 0.043). That said, neither difference remained statistically significant after adjusting for multiplicity (Padjusted ≥ 0.054). No differences in 30:15 ratio or standing systolic blood pressure were observed with foot immersion alone, while heart rate variability and baroreflex sensitivity were unaffected by either cooling intervention. While foot immersion with neck cooling potentially improved cardiac autonomic responses in older adults exposed to simulated indoor overheating, these effects were small and of questionable clinical importance.

Distal convoluted tubule-specific disruption of the COP9 signalosome but not its regulatory target cullin 3 causes tubular injury.

The disease familial hyperkalemic hypertension (FHHt; also known as Gordon syndrome) is caused by aberrant accumulation of with-no-lysine kinase (WNK4) activating the NaCl cotransporter (NCC) in the distal convoluted tubule (DCT) of the kidney. Mutations in cullin 3 (CUL3) cause FHHt by disrupting interaction with the deneddylase COP9 signalosome (CSN). Deletion of Cul3 or Jab1 (the catalytically active CSN subunit) along the entire nephron causes a partial FHHt phenotype with activation of the WNK4-STE20/SPS1-related proline/alanine-rich kinase (SPAK)-NCC pathway. However, progressive kidney injury likely prevents hypertension, hyperkalemia, and hyperchloremic metabolic acidosis associated with FHHt. We hypothesized that DCT-specific deletion would more closely model the disease. We used Slc12a3-Cre-ERT2 mice to delete Cul3 (DCT-Cul3-/-) or Jab1 (DCT-Jab1-/-) only in the DCT and examined the mice after short- and long-term deletion. Short-term DCT-specific knockout of both Cul3 and Jab1 mice caused elevated WNK4, pSPAKS373, and pNCCT53 abundance. However, neither model demonstrated changes in plasma K+, Cl-, or total CO2, even though no injury was present. Long-term DCT-Jab1-/- mice showed significantly lower NCC and parvalbumin abundance and a higher abundance of kidney injury molecule-1, a marker of proximal tubule injury. No injury or reduction in NCC or parvalbumin was observed in long-term DCT-Cul3-/- mice. In summary, the prevention of injury outside the DCT did not lead to a complete FHHt phenotype despite activation of the WNK4-SPAK-NCC pathway, possibly due to insufficient NCC activation. Chronically, only DCT-Jab1-/- mice developed tubule injury and atrophy of the DCT, suggesting a direct JAB1 effect or dysregulation of other cullins as mechanisms for injury.NEW & NOTEWORTHY CUL3 degrades WNK4, which prevents activation of NCC in the DCT. CSN regulation of CUL3 is impaired in the disease FHHt, causing accumulation of WNK4. Short-term DCT-specific disruption of CUL3 or the CSN in mice resulted in activation of the WNK4-SPAK-NCC pathway but not hyperkalemic metabolic acidosis found in FHHt. Tubule injury was observed only after long-term CSN disruption. The data suggest that disruption of other cullins may be the cause for the injury.

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 a bout 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.

Markers of enterocyte damage, microbial translocation, and systemic inflammation following 9 h 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 years; 10 females) and 37 older (68-73 years; 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 = 0.241). 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.

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

Sodium-glucose cotransporter-2 inhibitors (SGLT2i) reduce blood pressure (BP) in patients with hypertension, 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-wk-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 an abundance of cleaved ENaC α and γ subunits compared with Wistar rats, none of which were affected by EMPA. Another set of male Wistar rats was treated with EMPA, the NCC inhibitor hydrochlorothiazide (HCTZ), and 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.NEW & NOTEWORTHY This study suggests that reduced NHE3-mediated sodium reabsorption in the renal proximal tubule may account, at least in part, for the BP-lowering effect of SGLT2 inhibitors in the setting of hypertension. It also demonstrates that chronic treatment with SGLT2 inhibitors upregulates NCC activity, phosphorylation, and expression in the distal tubule of normotensive but not hypertensive rats. SGLT2 inhibitor-mediated upregulation of NCC seems crucial to counteract proximal tubule natriuresis in subjects with normal BP.

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.

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.

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.

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.

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.

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.