Citations

Introduction: Inflammatory bowel disease is characterized by remitting and relapsing bouts of intestinal inflammation accompanied by sensory and motor dysfunction that persists beyond the active periods of inflammation. Evidence suggests that neural pathways of the Gut-Brain Axis, particularly the sensory and sympathetic nervous system, are involved in pathophysiology, but until recently technical restrictions have limited our understanding of the underlying neural circuit mechanisms. In this study, we examined how sensory-sympathetic circuits are modified by colitis and how these alterations contribute to dysfunction during both active inflammation and post-inflammatory recovery. Methods: E2a-GCaMP mice were treated with vehicle or 3% dextran sodium sulfate (DSS) for 5 days to induce experimental colitis and switched to normal drinking water. Mice were assessed at two time points: Day 7 (two days after ending DSS treatment), representing active inflammation, and Day 21 to represent recovery. Inflammation and mucosal damage were evaluated using disease activity index scores and hematoxylin and eosin (H&E) staining. Colon motility was assessed in vivo via colonic bead expulsion and ex vivo using whole-colon preparations. We performed Ca 2+ imaging of sympathetic motor neurons in prevertebral ganglia (PVG) and sensory neurons in dorsal root ganglia (DRG) neurons in ex vivo preparations that preserve extrinsic neural circuitry with the colon (intestinofugal-sympathetic and spinal cord-sympathetic pathways). Results: Histological analysis confirmed hallmark features of colitis, including colon shortening, immune infiltration, and mucosal damage at 7 days post-DSS (p< 0.05, n=3–4), which resolved after recovery. During active inflammation, in vivo colon transit time was significantly faster, and this persisted after recovery (p< 0.05, n=7-9). Ex vivo motility assays in isolated colons also revealed DSS-induced dysmotility during active inflammation, but the irregular colon motor complexes became normal after recovery (p< 0.05, n=7-9), suggesting that extrinsic neural pathways contribute to prolonged dysmotility. In preparations without the spinal cord, PVG neurons exhibited increased activity and increased responses to distension during active inflammation but returned to baseline after recovery (p< 0.05, n=3-4), suggesting alternative sensory-PVG pathways contribute to persistent dysmotility. In preparations with spinal circuits intact, thoracolumbar (TL), but not lumbosacral (LS), DRG sensory neurons were hypersensitive to colonic distension during inflammation (p< 0.05, n=8-9). Furthermore, TL spinal stimulation evoked more responses in PVG neurons during active inflammation, but this was associated with decreased inhibition of ENS motor circuits (p< 0.05, n=6-7) and increased Ca 2+ signaling in the epithelium (p< 0.05, n=5), suggesting shifts in TL spinal-sympathetic motor output. Preliminary studies show an increase in spontaneous activity of TL DRG neurons in recovery that does not occur at LS levels (p< 0.05, n=3-6), which suggests that thoracolumbar spinal pathways remain hyperexcitable after recovery.Conclusions: Our findings indicate that TL spinal pathways associated with sympathetic motor outflow contribute to pain and dysmotility during active inflammation and may contribute to the post-inflammatory recovery processes that seem to go awry in IBD patients with relapsing inflammation. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

~80% of kidney stones (KS) are composed of calcium oxalate and small increases in urinary oxalate significantly enhance the stone risk. We previously discovered Oxalobacter formigenes (Of)-derived secreted factors which stimulate oxalate transport by human intestinal Caco2-BBE (C2) cells and reduce urinary oxalate excretion in hyperoxaluric mice by enhancing colonic oxalate secretion (JASN, 2017). The small Sel1-like protein-derived peptides (P8 and P9) were identified as the major Of-derived factors (AJP Cell Physiol, 2023), acting via the oxalate transporters SLC26A6 (A6) and SLC26A2 (A2). P8/9-induced stimulation of oxalate transport in C2 cells is likely PKA-dependent based on studies using the PKA inhibitor H89. Enhancing gut oxalate secretion with subsequent fecal excretion represents a novel therapeutic pathway for getting rid of body oxalate. Building on these findings, the study objective is to test the hypothesis that the observed regulation is indeed PKA-mediated, through mechanisms potentially involving other signaling pathways and/or altering A6 total and/or surface protein expression. To provide further evidence that it is PKA-dependent, we successfully identified the binding mode of P8/9 bound to PKA using molecular docking (ZDOCK). The regulatory subunit of PKA binds two cyclic AMPs and P8/P9 are predicted to interact at the cyclic AMP binding site according to our PKA-peptide modeling studies. To directly confirm the role of PKA, we used siRNA to knockdown its α catalytic subunit (PKA-α) in C2 cells. PKA-α mRNA expression is reduced by >90% as assessed by qPCR. Importantly, siRNA knockdown of PKA-α completely abolished P8/P9-induced stimulation of 14C-oxalate uptake by C2 cells in pilot studies, confirming the role of PKA in P8/9-mediated stimulatory regulation of intestinal oxalate transport. To identify the signaling pathways potentially upstream and/or downstream of PKA we used relatively selective pharmacological inhibitors. Interestingly, the stimulatory effects of P9 on 14C-oxalate uptake by C2 cells are significantly blocked by the ERK1/2 inhibitor U0126 (~90%), the PI3K inhibitor LY294002 (~84%), and the p38 inhibitor SB 202190 (~73%), while the general tyrosine kinase inhibitor genistein has no effect. Inhibitors alone did not affect baseline transport. These results support the involvement of the MAPK ERK1/2, PI3K, and p38 in the signaling pathways regulating intestinal oxalate transport by P8/9. We used immunoblotting and surface biotinylation studies to evaluate whether P8/9 stimulates oxalate transport in C2 cells by increasing its total and/or surface protein expression. P8/9 peptides did not affect A6 total and surface protein expression levels in preliminary studies. We could not assess A2 total and surface protein expression due to lack of good working antibodies. In view of our previous findings that the observed regulation is due to P9-induced increase in Vmax and reduction in Km, and since P8/9 did not affect A6 surface protein expression, it is likely possible that the observed stimulation is due to mechanisms including P8/9-induced enhanced A6 transport activity resulting from an increase in the intrinsic activity of the preexisting A6 membrane transporters. We conclude that P8/9 stimulates intestinal oxalate transport in C2 cells via signaling pathways including PKA, ERK1/2, PI3K, and p38 and through mechanisms including P8/9-induced enhanced A6 transport activity. Having the P8/9-PKA bound conformation determined, we plan to optimize P8/P9 into small molecule like drugs with better stability and clinical translational potential in terms of stimulating intestinal oxalate secretion, leading to reduced plasma and urinary oxalate levels. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Background: Exogenous and endogenous polyamines are critical for gastrointestinal (GI) barrier integrity, of which putrescine, spermidine and spermine are most well studied. Our recent work shows n-acetylputrescine, a putrescine derivative, correlates with increased intestinal permeability and loss of microbial diversity. Despite this, bacterial mechanisms of n-acetylputrescine production or direct effects on barrier integrity are unknown. Aim: Determine the role of microbiota in shaping n-acetylputrescine abundance and the effect on barrier integrity. Methods: N-acetylputrescine effects on barrier were assessed both in vitro using Caco-2 and ex vivo in Ussing using colonic strips from conventional mice. Germ-free (GF) mice were monocolonized with wild-type E. coli (SK929) or E.coli SK930 (2-weeks, n=4mice/group), strains given by Dr. Shin Kurihara. E. coli SK930 is deficient in SpeC, SpeF and SpeB, genes generating putrescine, required for n-acetylputrescine production. Untargeted metabolomics (Metabolon) was performed on fecal pellets. Targeted isotope tracing with [13C6, 15N4]-arginine delivered via enema was done to track bacterial production of n-acetylputrescine by Fusobacterium varium, a known producer, in vivo with isotopolog labeling measured by LC/HRMS. Results: In vitro n-acetylputrescine (100mM) addition to Caco-2 monolayers caused significant barrier disruption compared to controls (%Baseline TER, 13.7±6.7 vs 102.8±9.4, 3h postexposure, n=3 p=0.0002), not seen at lower (10mM) concentration. N-acetylputrescine caused increased flux of 300Da fluorescein (12.6±2.0 vs 6.0±0.2 mg/mL, n=3 p=0.004) and 4kDa FITC-Dextran flux on Ussing compared to untreated controls (999±94 vs 562±121 ng/hr/cm2, n=3 p= 0.01). Mutant E. coli SK930 monocolonized mice had similar fecal n-acetylputrescine to GF animals. In contrast, wild type E. coli SK929 monocolonization led to significantly greater n-acetylputrescine (5-fold, FDR< 0.001) compared to SK930, indicating a bulk of n-acetylputrescine production is driven by bacterial mechanisms. Pathway enrichment analysis predicted decreased spermidine and spermine biosynthesis in SK930 monocolonization, indicative of impaired polyamine metabolism, compared to SK929 (FDR< 0.01). Targeted metabolomics on fecal pellets showed a shift in the production of isotopolog [13C1, 15N0]-N-acetylputrescine to increased isotopolog [13C4, 15N2]-N-acetylputrescine concentration after monocolonization with F. varium compared to GF mice (5.9±5.0 vs. 2.1±3.6 pg/mg stool, n=3mice/group p=0.05) suggesting microbiota driven shifts in n-acetylputrescine metabolism. Conclusion: Luminal n-acetylputrescine production and profile is driven by bacteria in the GI tract. Concentration dependent effects of n-acetylputrescine suggest insufficient regulation by intestinal microbiota can lead to accumulation of n-acetylputrescine and the intestinal barrier disruption seen in human studies. Ongoing studies aim to identify bacterial taxa critical for n-acetylputrescine suppression, the mechanisms involved, and impact on GI health. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Background: As part of the bidirectional gut-brain communication system that influences gastrointestinal function, vagal pathways coordinate with the activity of interstitial cells of Cajal (ICC) to regulate gastric motility. Impairments in ICC slow wave activity and altered vagal tone have been implicated in diabetic gastroparesis and obesity, but technical limitations have restricted our ability to measure integrated and bidirectional activity in vagal-ICC pathways. Furthermore, the influence of physiological and meal-induced stimuli like gastric distension on these pathways is not well understood. We aimed to develop a calcium imaging approach in ex vivo nodose ganglion-vagus nerve-stomach preparations to investigate vagal-ICC pathways and test the effects of gastric distension on vagal activation, ICC network activity, and their functional interactions. Methods: Calcium imaging was performed in ex vivo preparations, in which the stomach and vagus nerves (with or without the ganglion) remain intact. EIIa-GCaMP6 (n=3) and cKit-GCaMP6 (n=3) mice were used to image Ca 2 + signals from both vagal neurons and ICC or ICC alone, respectively. Two-minute recordings were captured in the following stomach conditions: (1) Undistended; (2) Undistended + vagal nerve stimulation (VNS, 20 Hz, 1 s); (3) Distended (0.25 ml volume distension); (4) Distended + VNS. Ca 2 + signals in ICC and neurons, along with tissue displacement, were analyzed using Image J. Data from both GCaMP models were pooled for ICC. Results: Acute gastric distension significantly activated vagal sensory neurons in nodose ganglion compared to baseline (p=0.0193), and VNS evoked Ca 2 + responses in ICC and neurons, confirming intact nerve pathways. Both VNS and gastric distension altered the direction of ICC propagation, significantly increasing retrograde waves (p=0.0025 and p=0.0032, respectively), and VNS had no effect on retrograde waves in distended stomachs. VNS and distension also reduced length of propagation of anterograde ICC waves (p=0.019 and p=0.0004, respectively), and again, VNS effects were not observed during distension. VNS increased the velocity of ICC wave propagation, but only in undistended stomach (p=0.0283). Finally, VNS increased ICC pacemaker frequency regardless of the distension state (p=0.0123). Conclusion: Both acute gastric distension and VNS altered ICC network activity, with similar effects on network parameters. However, most of the VNS effects on ICC were blocked during distension. VNS increased the frequency of ICC network waves in both undistended and distended stomachs, suggesting that VNS can influence ICC pacemakers regardless of distension state. These findings have important implications for designing therapeutic strategies for gastroparesis. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Introduction/Aim: Heart failure (HF) is a complex clinical syndrome, characterised by reduced exercise capacity (VO 2 peak) and quality of life leading to premature death. The reduction in VO 2 peak is a key prognostic indicator, and improving exercise capacity remains a critical therapeutic target. In athletes, the use of intermittent hypoxia training to improve VO 2 peak and performance is well established. Changes in VO 2 peak are typically accompanied by improved oxygen carrying capacity with increased haemoglobin (Hb) concentration mediated through increased erythropoietin (EPO) production in the kidney. The purpose of this pilot study was to examine the feasibility of using intermittent hypoxia training in individuals with HF, and examine changes in VO 2 peak, EPO and Hb. We hypothesised that intermittent hypoxia training would be feasible, well tolerated, and improve VO 2 peak, EPO production, Hb concentration in HF with reduced ejection fraction (HFrEF). Methods: Ten male participants with stable, optimally managed HFrEF (Age: 73 ± 5 yr; Ejection Fraction: 37 ± 11%) participated in the study. The training stimulus consisted of 12 x 60-minute sessions of intermittent hypoxia over a 2-week period. During each 60-minute session participants received 6 x 5 minutes of pulsed, reduced FIO 2 exposures interspersed with 5 min of normoxia (total of 30 min hypoxic exposure per session). All participants commenced at 18% FIO 2 , which was decreased to 12% by the 3-4th sesion, as tolerated. Throughout each exposure, oxygen saturation (SpO 2 ), heart rate, blood pressure and dyspnoea (modified Borg scale) were monitored every 5 min. Changes in VO 2 peak were measured using a cycle ergometry, cardiopulmonary exercise test. Changes in VO 2 peak, N-terminal pro B-type natriuretic peptide (NT-proBNP), EPO and Hb concentration were measured pre and immediately post intermittent hypoxic training. Results: All participants completed between 10-12 (median n=12) sessions over the 2-week period. Intermittent hypoxia was well-tolerated with no adverse events. The mean SpO 2 during the hypoxic exposure was 88 ± 4%, and median reported dyspnoea 0 (range 0-1). Following 2 weeks of intermittent hypoxic training, there were significant increases in VO 2 peak (mean difference[MD]: +1.18 mL·kg - ¹·min - ¹, 95% CI 0.47 to 1.89, p = 0.010); EPO (MD: +3.33 IU/L, 95% CI 1.35 to 5.32, p = 0.011); with a modest increase in Hb concentration that approached significance (MD: +4.00 g/L, 95% CI 0.51 to 7.49, p = 0.055). NT-proBNP remained unchanged (MD: –107 pg/mL, 95% CI –356 to 142, p = 0.424). Conclusion The results of this pilot study suggest that in clinically stable HFrEF, 2 weeks of intermittent hypoxic training results in a significant improvement in VO 2 peak, accompanied by increases in EPO and potentially levels of Hb. In our participants, this 60-minute pulsed intermittent hypoxic protocol was extremely well tolerated without any adverse effects, and may improve oxygen carrying capacity of the blood in HFrEF. Further research on the mechanisms of adaptation and the translation of the response into different HF phenotypes is warranted. Supported By: The Prince Charles Hospital Foundation Innovation Grant INN202-049 and Griffith University. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Staphylococcus aureus is a well-known pathogen responsible for a wide range of diseases with life-threatening consequences. Its public health burden continues to be significant due to pervasive colonization and growing antimicrobial-resistance (i.e., methicillin-resistant S. aureus, MRSA). In the present study we found that exposure of human bronchial epithelial (hBE) cells to spent culture supernatants of the MRSA strain USA300 induced an increase in mitochondrial ROS production and ATP release that was inhibited by pretreatment with the ROS scavenger glutathione (GSH). ATP secretion involved activation of probenecid-sensitive pannexin 1 channels and bafilomycin A1-dependent exocytosis. ATP exocytosis was inhibited by an antagonist peptide targeting the Receptor for Advanced Glycation End (RAGE) products suggesting that RAGE activation was involved in regulating vesicular ATP release. ATP secretion produced a sustained increase in intracellular Ca 2+ concentration which was dependent upon Ca 2+ uptake from the extracellular solution. Moreover, the sustained JE2-induced Ca 2+ response was not observed in spent supernatants from the less virulent S. aureus agrA mutants used to stimulate hBE cells whereas media from fadX and codY mutants produced responses similar to JE2. Furthermore, wildtype, fadX and codY supernatants evoked secretion of the type-2 inflammatory cytokine IL-33, whereas no response was elicited by media from the less virulent argA mutant. These findings indicate that exposure to secreted virulence factors from S. aureus induces oxidative stress in hBE cells that leads to ATP secretion, sustained Ca 2+ uptake and IL-33 secretion, which has been previously shown to induce type-2 inflammation in airways. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Background: During hypoxic exercise, oxygen transport becomes increasingly challenged as oxygen availability decreases. As the inspired oxygen fraction (FiO 2 ) decreases, peripheral oxygen saturation (SpO 2 ) gradually declines, then drops rapidly upon reaching the steepest point of the sigmoidal shaped, oxyhemoglobin dissociation curve. This reduction in FiO 2 creates a distinct SpO 2 “breakpoint,” which likely represents the threshold at which compensatory mechanisms, such as increases in ventilation and cardiac output, can no longer maintain arterial oxygenation. Several physiological factors may modulate this breakpoint, including hemoglobin concentration, blood volume, and ventilatory capacity. This study aimed to determine the SpO 2 breakpoint during steady-state, submaximal, hypoxic exercise and examine whether factors like sex, age, and fitness level influence the SpO 2 breakpoint. We hypothesized that SpO 2 breakpoints would differ by sex, age, and fitness level, with earlier breakpoints visible in females, older adults, and those with higher aerobic fitness. Methods: Twenty-six physically active adults (15M: age 35±9 yrs, [Formula: see text]O 2 max 54.3±15.5 mL/kg/min, 11F: age 38±10 yrs, [Formula: see text]O 2 max 44.6±5.2 mL/kg/min) participated in a two-day study. Day one: participants performed a treadmill [Formula: see text]O 2 max test. Day two: participants walked at a steady state of ~50% of their [Formula: see text]O 2 max, for 40 minutes, while FiO 2 was progressively reduced every 5 minutes in a stepwise fashion (0.25, 0.21, 0.19, 0.17, 0.15, 0.13, 0.12, 0.11). The test was terminated when SpO 2 dropped below 70%. SpO 2 was measured continuously using a clinical-grade oximeter. Analysis was performed in RStudio by first determining each participant’s individual SpO 2 vs time breakpoint. Male and Female breakpoints were compared to assess sex-based differences. To evaluate whether SpO 2 breakpoint timing differed by age or fitness level, participants were then divided into tertiles for each variable. Age tertiles were derived from the sample distribution of chronological age, and fitness tertiles were defined using [Formula: see text]O 2 max (L/min). Mean breakpoint times were compared between the highest and lowest tertiles for both age and fitness. Mean SpO 2 breakpoints were compared between groups using independent sample t-tests. Significance was set to p< 0.05. Results: The overall mean breakpoint occurred at an SpO 2 of 90.7±2.8% during the 0.17 FiO 2 stage. No significant sex differences were observed in breakpoint SpO 2 and timing (M: SpO 2 = 90.7±2.7%, time = 18.6 min; F: SpO 2 = 90.7±3.0%, time = 17.7 min; p = 0.961). Age did not significantly influence breakpoint (younger tertile [21-30 yrs] SpO 2 = 91.0±2.4%, time = 18.4 min; older tertile [41-60 yrs] SpO 2 = 91.1±2.4%, time = 17.2 min, p = 0.953). Additionally, fitness level showed no significant difference on breakpoint (low fitness [1.91-3.01 L/min]: SpO 2 = 90.3±3.1%, time = 17.8 min; high fitness [4.38-5.25 L/min]: SpO 2 = 90.4±2.9%, time = 19.2 min; p = 0.992). Conclusion: During a hypoxic, steady state walking exercise, at ~50% [Formula: see text]O 2 max, we observed no difference in the peripheral oxygen saturation breakpoint, across sex, age, and fitness level. This may suggest that moderate exercise in hypoxia does not sufficiently challenge the cardiopulmonary system for individual variations in oxygen transport to influence the breakpoint. These findings imply that the SpO 2 breakpoint, during hypoxic exercise, may represent a physiological marker of the upper limit for maintaining oxygen saturation in healthy adults. Applying this protocol to identify SpO 2 breakpoints may offer insights for improving pharmacological interventions and our understanding of clinical populations with impaired oxygen transport. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Neuromotor control of sighs consists of two phases: Phase I, which resembles a preceding eupneic breath, and Phase II, an extra inspiration superimposed on Phase I. The diaphragm muscle (DIAm)—the primary inspiratory muscle in mammals—is activated during multiple behaviors, including sighs, and its activity can be quantified using electromyography (EMG). Although the biphasic structure of sighs has been described using airflow and pressure signals, how these phases are represented in DIAm EMG activity—and the underlying dynamics of DIAm motor unit (MU) recruitment—remains unclear. Previously we described that eupnea consists of a period of MU recruitment followed by a period of sustained MU activity before derecruitment. We hypothesize that during sighs Phase I follows a similar trajectory of MU recruitment as eupnea while Phase II reflects superimposed additional MU recruitment. DIAm EMG was recorded using fine wire electrodes in awake Sprague-Dawley rats (n=6) of both sexes. To determine the period of DIAm MU recruitment during both Phase I and Phase II, a stationarity analysis was performed. The stationarity analysis uses a reverse arrangement test applied to sequential, non-overlapping 10-ms bins across a longer analysis window. Because DIAm MU discharge rates rarely exceed 100 Hz, it is unlikely that a single MU fires more than once within each 10-ms window; thus, non-stationarity reflects MU recruitment rather than changes in discharge rate. An average of 17 ± 4 sighs per animal were identified (range: 10-20), with a frequency of 2.8 ± 0.1 min-1. Sighs had a burst duration of 1300 ± 405 ms, much longer than the burst duration of eupneic breaths (~598 ± 111 ms). During sighs there were two distinct periods of MU recruitment reflected by non-stationarity of the EMG signal. The MU recruitment duration of Phase I was significantly shorter than Phase II (100 ± 28 ms vs. 179 ± 53 ms, p = 0.041). The onset-to-peak duration during sighs averaged 489 ± 135 ms, with the duration of Phase I lasting longer than Phase II (298 ± 119 ms vs 179 ± 56 ms). It is important to note that Phase II is characterized by rapid, continuous recruitment throughout its duration. In contrast, Phase I had a longer overall duration, although the period of MU recruitment was relatively brief—similar to eupneic breathing. Using EMG stationarity in awake animals allowed us to characterize these underlying MU patterns without altering ventilation or disrupting the intact of the respiratory system. These results indicate that sighs reflect a distinct pattern generator superimposed on that generating eupneic breathing. It remains unclear whether sighs are triggered (e.g., reflexes) or spontaneously generated. Funding: NIH NHLBI 5R01HL146114 and NIA 5R01AG044615 This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Aged heterozygous endothelial nitric oxide synthase knockout mice (eNOS+/−) represent an important model of spontaneous cerebral small vessel disease. However, the role of endothelium in vasomotor function of cerebral arteries of aged eNOS+/− mice has not been characterized. In the process of generating endothelial specific β-site amyloid precursor protein cleaving enzyme-1 (BACE1)/eNOS+/− knockout mice, we performed control studies in 18 months old male eNOS+/− mice and their wild-type littermates generated on floxed C57BL/6-Bace1tm1.1mrl background. Vasomotor function of isolated basilar arteries was studied in vitro under pressurized conditions using a video dimensions analyzer. Contractions to acetylcholine were observed in aged eNOS+/− mice (19±13%), while acetylcholine caused endothelium-dependent relaxations in age-matched wild-type littermates (-17±7%, P< 0.05; n=9 per group). In contrast, endothelium-independent relaxations to NO-donor diethylamine-NONOate were unaltered in both wild-type littermates (-83±10%) and eNOS+/− mice (-90±4%; n=6 per group; P >0.05). Furthermore, contractions to U46619 were significantly augmented in eNOS+/− mice (63±3%; P< 0.05 vs. wild-type: 47±4%; n=6). Morphological analysis revealed that lumen diameter of basilar arteries was not different between wild-type (169±4 µm) and eNOS+/− mice (165±4 µm; P >0.05; n=9). Furthermore, blood glucose levels were comparable between wild-type (185±7 mg/dL) and eNOS+/− mice (183±12 mg/dL; P >0.05; n=10). Measurement of lipid profile showed that plasma cholesterol, HDL as well as triglyceride levels were unchanged in eNOS+/− mice (P >0.05 vs. wild-type; n=10). Interestingly, mRNA expression of BACE1 is significantly increased in cerebral arteries of aged eNOS+/− mice (0.0295±0.0009) as compared to wild-type littermates (0.0243±0.0017; P< 0.05; n=5 per group). Our results demonstrate that heterozygous eNOS deficiency exacerbates endothelial dysfunction in aged eNOS+/− mice and is accompanied by the imbalance between vasodilation and vasoconstriction of cerebral arteries. Endothelial dysfunction in aged eNOS+/− mice appears to be independent of alterations in systemic metabolic parameters. Notably, partial deficiency of eNOS increases expression of BACE1, an enzyme responsible for production of amyloid-β peptides. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

We previously showed that the Drosophila Malpighian tubule (MT) can model the formation of calcium oxalate (CaOx) kidney stones. Two epithelial cell types, stellate cells (SC) and principal cells (PC) comprise MTs, with each transporting different solutes from the hemolymph to MT lumen. Prestin, Neat and Ndae1 contribute to transepithelial ox2-, HCO 3 - transport and fluid secretion. Neat (2HCO 3 - /ox2- exchange) and NDAE1 (Na + ,2HCO 3 - /Cl - exchange) functionally combine to takeup hemolymph ox2-, can also facilitate HCO 3 - uptake. Prestin then secretes ox2- via Cl - / ox2-exchange to create luminal MT CaOx crystals, but it can also exchange Cl - /HCO 3 - . This study examines transepithelial ion secretion by Prestin (Slc26a6, apical), Neat (Slc26a1-like, basal) and Ndae1 (Slc4a8-like, basal). We use a functionally similar, simple genetic model (Drosophila Malpighian tubules, MT) to measure renal CaOx crystallization (NaOx feeding), membrane transport (pHi, fluorescent pH sensors) and fluid secretion (Ramsey assay) to: (1) compare cell-specific gene knockdown (KD) vs gene knockout, (2) quantify CaOx crystallization and intracellular pH (pHi) regulation by CO 2 /HCO 3 - , and (3) humanize these transporters by replacing them with human homologs. We hypothesize that humanizing individual transporters will restore MT physiology. Candidate transporter genes are tested for transepithelial ox2-and HCO 3 - secretion with gene-specific knockdown or knockout, using CRIMIC (CRISPR-Mediated Integration Cassette) alleles, in MTs due to comparable function to mammalian nephrons. Neat and NDAE1 work together to take-up hemolymph ox2- or HCO 3 - then Prestin secretes ox2- or HCO 3 - into the MT lumen. Prestin, Neat or NDAE1 knockdowns in principal cells decrease CaOx crystals. NDAE1 but not Neat knockdown in SC increases CaOx crystals like AQP (Drip, Prip)-knockdowns while either KD enhance HCO 3 - transport. Ndae1-SC-KD reduces fluid secretion like AQP-KD. Each CRIMIC allele must be physiologically compared to PC/SC-KDs because Ndae1-KO but not Neat-KO mirrors PC rather than SC function. Ndae1-SC-KD shows that HCO 3 - and Cl - secretion cause fluid secretion. Human homolog replacements are on-going. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.