Physiological Reports

BackgroundHypertension, the persistent elevation of blood pressure (BP), is characterized by chronic low-grade inflammation and systemic cytokine release. Circulating cytokines contribute to the development of hypertension and end-organ damage. However, the specific immune profile associated with the progression of hypertension remains unclear. We hypothesize that a plasma cytokine signature reflects early BP changes in older adults. MethodsSeventy participants aged 57-81 years were categorized as normotensive (n = 17), elevated BP (n = 10), or hypertensive (n = 43) based on 24-hour ambulatory BP monitoring and antihypertensive treatment status. Plasma IL-1{beta}, IL-6, IL-10, IL-17A, IL-21, IL-22, IL-23, and TNF- were quantified using immunoassays. Partial Pearson correlations adjusted for demographic and biochemical covariates were used to assess associations between cytokines, BP, and cytokine-cytokine networks. ResultsIn untreated hypertensive individuals, plasma IL-23 was positively correlated with 24-hour diastolic BP. Antihypertensive treatment was associated with reduced IL-17A concentrations, which are negatively associated with 24-hour systolic BP. In the elevated BP group, IL-21 concentrations were higher than in normotensive individuals. To further characterize the cytokine signature, cytokine-cytokine correlations were examined. IL-23 and IL-17A were positively correlated with most interleukins, whereas TNF- showed few associations. IL-1{beta} exhibited strong correlations with both IL-23 and IL-17A, particularly in untreated participants. ConclusionIL-23 and IL-17A are associated with BP status and are broadly interconnected with other inflammatory cytokines, highlighting the potential importance of the IL-23/IL-17A axis in the hypertension of development. Early alterations in IL-21 in elevated BP may reflect immune changes that precede the onset of hypertension.

Globally, hypercholesterolemia affects over 20% of the population; and while many studies have examined its impact on cardiovascular health, little is known about its effects on the lymphatic system. In mice, hypercholesterolemia has been linked to multiple aspects of lymphatic dysfunction; and a recent study demonstrated that cholesterol depletion by cyclodextrins promoted lymphatic vessel regeneration and restored lymphatic drainage in mouse models of lymphedema. Collecting lymphatic vessels rely on the spontaneous and highly entrained contractions of lymphatic muscle cells (LMCs) and competent unidirectional on-way valves to propel lymph forward. Critical to lymphatic pacemaking and contractility is the proper functioning of ion channels, which are known to be modulated by the cholesterol content in the plasma membrane. Therefore, we sought to understand the role cholesterol plays in regulating lymphatic contractility. The effects of cholesterol depletion by the cyclodextrins M{beta}CD and HP{beta}CD were assessed in cannulated and pressurized inguinal-axillary collecting lymphatic vessels (CLVs) from C57BL6/J (WT) mice. Noteworthy, studies have shown that HP{beta}CD is safe for human use, and in fact, it is commonly used as a drug excipient. Acute treatment with both cyclodextrins significantly increased the pumping capacity of CLVs, as demonstrated by the increased contraction amplitudes by [~]50{+/-}12% and calculated fluid volume displacement by each contraction by [~]35{+/-}11%. Calcium imaging demonstrated that HP{beta}CD increased the amplitude and duration of the large Cav1.2-mediated calcium events (termed calcium flashes. In contrast, cholesterol supplementation by incubation with BODIPY-cholesterol, which presumably incorporates cholesterol into the cell membrane, significantly impaired the contractile activity of CLVs compared to controls by decreasing contraction amplitude (control: 42{+/-}2 {micro}m versus BODIPY-cholesterol: 20{+/-}7{micro}m) and calculated fluid volume displacement (control: 9.2{+/-}3.9nL versus BODIPY cholesterol: 3.3{+/-}1.2nL) which were significantly restored with subsequent cholesterol depletion using HP{beta}CD (amplitude: 36{+/-}11{micro}m, volume displacement: 5.5{+/-}2.4nL). Similarly, treatment with HP{beta}CD significantly improved the contractile capacity of dysfunctional CLVs isolated from hypercholesterolemic ApoEKO mice. In conclusion, changes to cell membrane cholesterol content acutely and significantly altered CLV contractility with depletion improving contractility associated with recruitment of voltage-gated Cav1.2 channels in lymphatic muscle cells (LMCs). Future studies from our lab will determine whether pharmacological depletion of membrane cholesterol can be therapeutic strategy to improve and/or restore lymphatic contractile function in secondary lymphedema, including obesity/hypercholesterolemia-induced and cancer-related lymphedemas.

Left ventricular hypertrophy, common in cardiometabolic and renal disease, is a major risk factor for cardiovascular morbidity and mortality. Left ventricular mass is a highly heritable, polygenic trait. Linkage studies in WKY and SHRSP rats have identified a quantitative trait locus for left ventricular mass index on chromosome 14. Congenic strains, where trait-associated genetic loci are introduced into a control strain, can identify causal genetic mediators relevant to human disease. Chromosome 14 congenic (WKY.SPGla14a), WKY, and SHRSP strains underwent cardiac phenotyping and transcriptome profiling at; 1-3 days (neonate), 5 weeks, and 16-weeks. Compared to WKY, LVMI was significantly increased in SHRSP and WKY.SPGla14a at 5 weeks (LVMISHRSP-WKY=0.26g/kg, LVMIWKY.SPGla14a-WKY=0.30g/kg), prior to measured hypertension in this model. SHRSP blood pressure was significantly greater than WKY.SPGla14a, and WKY from 12-20 weeks (AUCdiff=497 vs WKY, AUCdiff=412 vs WKY.SPGla14a). Cardiac transcriptome analysis of neonate, 5-week, and 16-week hearts identified significantly increased expression of secreted phosphoprotein 1 (Spp1/osteopontin) in SHRSP and WKY.SPGla14a compared to WKY, which is positioned within the transferred congenic region. Overexpression of Spp1 mRNA significantly increased H9c2 cell size and was shown to be transferred in small extracellular vesicles (sEV). Overexpression of Spp1 in neonatal chromosome 14 congenic and SHRSP strains preceded development of increased cardiac mass and onset of hypertension. The congenic strategy identified Spp1 as a positional and functional candidate gene determining increased LVMI in the SHRSP model of human cardiovascular disease.

Background Limited heart rate rise contributes to reduced exercise tolerance for people who have heart failure with reduced ejection fraction (HFrEF), yet rate-adaptive pacing does not improve functional capacity due to an attenuated force-frequency relationship (FFR). How the FFR relates to total peripheral resistance and sympathetic tone in HFrEF is unknown. Methods In a prospective, observational study, participants with HFrEF and controls underwent an incremental pacing protocol, during which heart rate was increased from 50 to 140 beats per minute. At each heart rate increment LV contractility was measured by echocardiography to determine the FFR, as well as continuous beat-to-beat measurement of systolic and diastolic blood pressures with a plethysmography device to determine cardiac output, total peripheral resistance and blood pressure variability (BPV). A microneurography study was then conducted to measure muscle sympathetic nerve activity (MSNA) during incremental pacing. Results A total of 157 participants with HFrEF and 55 controls (mean age 71.1{+/-}1.4 years, 172 (81.1%) male) underwent the pacing protocol. We observed single units in seven of 11 participants who participated in the microneurography study. In both groups, LV contractility and cardiac output increased until the peak of the FFR, after which these declined. We observed a reduction in total peripheral resistance, blood pressure variability, MSNA frequency and incidence coinciding with the peak of the FFR, beyond which these increased. Whilst these relationships were present in both groups, they were more evident in participants with HFrEF. Conclusions For people with HFrEF there is a bidirectional relationship between heart rate and sympathetic activation, with a nadir of sympathetic tone occurring at the peak of the FFR. Both excessively low and high heart rates are accompanied by greater sympathetic activation. Taken together, these data suggest that optimal heart rate targets for HFrEF are likely to be individual.

BackgroundHydrogen sulfide (H2S) is an important endothelial-derived vasodilator, but the signaling mechanism remains incompletely understood. We previously demonstrated that H2S-mediated vasodilation requires transient receptor potential vanilloid type 4 (TRPV4) channels. Because H2S has been reported to enhance heme oxygenase (HO) activity and HO-derived carbon monoxide (CO) regulates endothelial signaling, we hypothesized that H2S-mediated vasodilation requires HO-2-derived CO. MethodsPressure myography was performed in isolated rat mesenteric arteries to determine the contribution of HO, TRPV4, eBK, and SK/IK channels to H2S-mediated vasodilation. HO-2 sulfhydration was assessed using a maleimide assay, and spatial association among HO-2 and TRPV4 was examined using proximity ligation assays in human aortic endothelial cells. ResultsH2S Selicited concentration-dependent vasodilation that was abolished by HO inhibition. Repletion of CO restored H2S-mediated vasodilation in the presence of HO inhibition. CO-mediated vasodilation was abolished by TRPV4 and SK/IK inhibition but was unaffected by eBK inhibition. H2S increased HO-2 sulfhydration and enhanced HO activity. In endothelial cells, HO-2 and TRPV4 exhibited close spatial association. ConclusionsThese findings support a model in which H2S stimulates HO-2-derived CO production, leading to TRPV4-dependent endothelial signaling, SK/IK activation, and vasodilation. Together, the data support the existence of an endothelial HO-2/TRPV4/SK/IK signaling domain that contributes to H2S-mediated vascular reactivity.

Background: Heart failure (HF) is classified by ejection fraction: reduced EF (<40%) is HFrEF and preserved EF (>50%) is HFpEF. Unlike HFrEF, no therapeutic agent improves mortality in HFpEF. The molecular mechanism that produces HFpEF is not completely understood, but the cascade of pathology that produces HFpEF is thought to begin with changes in vascular reactivity, including a decrease in NO mediated vasodilatation, which coupled with subsequent changes in contractility, energetics and coronary blood flow produce HFpEF. If abnormal vascular reactivity is the initial step in the pathological cascade that produces HFpEF, restoring and/or improving vascular reactivity could represent a novel treatment strategy. Vascular reactivity is primarily regulated by myosin light chain phosphatase, which has catalytic, myosin targeting (MYPT1) and 20kDa subunits. Alternative mRNA splicing of exon24 (E24) of the MYPT1 transcript produces MYPT1 isoforms that differ by the presence or absence of a COOH-terminal leucine zipper (LZ+/LZ-); E24 exclusion produces an NO responsive LZ+ MYPT1, while E24 inclusion produces an NO unresponsive LZ- MYPT. Methods: We used the mouse two-hit model of HFpEF (high fat diet and L-NAME) and treated mice with an antisense octo-guanidine targeting the 5' splice site of E24 (ASO-E24) to increase the expression of the NO responsive, LZ+ MYPT1 isoform in vascular smooth muscle. Invasive and noninvasive hemodynamics were used to determine LV function. Results: Compared to mice with HFpEF, ASO-E24 treatment maintains LZ+ MYPT1 expression (4.7{+/-}0.7au v 1.0{+/-}0.4au v 2.0{+/-}0.4au, control v HFpEF v ASO-E24 Rx, p<0.05), improves diastolic function; LVEDP (10{+/-}1mmHg v 20{+/-}4mmHg v 14{+/-}3mmHg, p<0.05), dP/dtmin (-8000{+/-}300mmHg/s v 6000{+/-}500mmHg/s v 8500{+/-}700mmHg/s, p<0.05), both early (E; 0.60{+/-}0.05m/s v 0.42{+/-}0.06m/s v 0.64{+/-}0.06m/s, p<0.05) and late diastolic filling (A; 0.38{+/-}0.03m/s v 0.24{+/-}0.02m/s v 0.47{+/-}0.04m/s, p<0.050 and also prevents the increase in lung weight (167{+/-}5g v 175{+/-}7g v 166{+/-}5g, p<0.05). Further, mice treated with ASO-E24 maintained normal relaxation to 8Br-cGMP (65{+/-}5% v 44{+/-}9% v 72{+/-}9%, p=0.05). Conclusion: These data demonstrate that maintaining normal LZ+ MYPT1 expression and vascular reactivity prevent the development of HFpEF. These results are consistent with the hypothesis that abnormal vascular reactivity is the initial and primary step in the pathological cascade that produces HFpEF and ASO-E24, which is designed to preserve normal LZ+ MYPT1 expression and vascular reactivity, could represent a novel and effective treatment strategy for HFpEF.

BackgroundInsular damage leads to marked cardiovascular alterations and the mechanisms need to be understood. Mouse models provide unique opportunities to gain insights into pathophysiological mechanisms. Here, we evaluated the effects of rilmenidine, a centrally acting antihypertensive drug, on the cardiac functional parameters and cardiac inflammatory cell infiltration in a newly developed mice model of insular hemorrhagic stroke. MethodsC57BL/6J mice were instrumented for injection of blood or vehicle into the insular cortex (IC). Immediately after IC stroke induction, separate groups received intraperitoneal treatment with vehicle (0.9% NaCl, 0.1 mL/100 g) or rilmenidine (10 g/kg) for three days. Electrocardiogram recording,cardiac catecholamine levels and myocardial accumulation of immune cells were evaluated. ResultsMice subjected to hemorrhagic stroke exhibited higher baseline heart rate (HR) (control: 296 {+/-} 33 bpm vs. stroke: 349 {+/-} 38 bpm; P < 0.01) and prolonged QTc interval (control: 89 {+/-} 11 ms vs. stroke: 100 {+/-} 7 ms; P < 0.01). Stroke also increased cardiac norepinephrine levels (control: 9 {+/-} 4 ng/mg vs. stroke: 25 {+/-} 14 ng/mg; P < 0.05), as well as the number of myocardial CD68+ macrophages (control: 7 {+/-} 4 vs. stroke: 16 {+/-} 6 cells/field; P < 0.0001) and Ly6G+ neutrophils (control: 0.5 {+/-} 0.7 vs. stroke: 1.5 {+/-} 1 cells/field; P < 0.001). Rilmenidine treatment markedly prevented all major stroke- induced myocardial functional and inflammatory changes ConclusionsInsular hemorrhagic stroke in mice induces centrally mediated cardiac noradrenergic hyperactivation accompanied by myocardial accumulation of immune cells. These findings support the relevance of this murine model for investigating mechanisms associated with insular stroke.

Coronary flow waveforms have a distinct diastolic-dominant shape with periods of low or retrograde flow during systole. While the general waveform shape has been attributed to complex interactions between cardiac and vascular mechanics, there is limited research into the variability in coronary flow waveforms and what this variability may reveal about cardiac function. This work presents a shape analysis of left anterior descending artery (LAD) flow waveforms using Fourier transforms and Singular Value Decomposition (SVD) performed on baseline data collected from 32 pigs. Pigs included in the study reflect two breeds (Ossabaw and Yorkshire) and three different experimental conditions (lean-control, lean-paced, and obese-paced). Fourier transforms were used to decompose the waveforms into 15 harmonics for each pig. An SVD analysis is then used to extract temporal patterns of the waveforms. Correlations between pig-specific coefficients for the SVD modes and clinical metrics were used to investigate physiological explanations of LAD waveform variability. Temporal LAD flow patterns of the second SVD mode are significantly correlated with heart rate. The third SVD mode significantly correlates with mean blood pressure and maximum hyperemic flow. Furthermore, the fourth SVD mode is weakly correlated with left-ventricular end diastolic pressure and endocardial-epicardial flow ratios. This work demonstrates that LAD flow waveforms can be broken down into temporal patterns that correlate with physiological features. Furthermore, this shape-analysis method allows for waveform reconstruction and simplifies visualization of the temporal patterns identified using SVD, an advantage over existing methods that focus on characterizing flow waveforms by points of interest.

Modulation of renal nerves to improve blood pressure (BP) control has become a topic of intense investigation over the last 10-15 years. Given that renal innervation is composed of mixed nerve fibers containing both afferent (sensory) and efferent (sympathetic) fibers, subsequent preclinical studies have been investigating their respective roles in hypertension pathobiology in different genetic and salt-sensitive rat models. Here we set out to investigate how renal afferent and efferent nerves regulate hypertension development in the chronic mild stress model (CMS). We show that in male CMS rats, ablation of afferent renal nerves (ARDNx) and all renal nerves (TRDNx) resulted in similar BP (104 {+/-} 2 mmHg vs. 101 {+/-} 3 mmHg, respectively), both reduced compared to the SHAM group (118 {+/-} 1 mmHg, p = 0.003 and p < 0.001, respectively) arguing for a prominent role of afferent renal nerves in CMS hypertension. Additionally, we show a reduction of vasopressin (AVP) V1b but not V1a receptor abundance in ARDNx CMS males but not females, suggesting that afferent renal nerves are involved in increase in BP via V1b AVP receptor. We additionally show that despite normal BP, female CMS rats display increased renal sympathetic nerve activity (RSNA; 2.39 {+/-} 0.23 bursts/beat vs. 1.44 {+/-} 0.12 bursts/beat, p < 0.005) measured directly with implanted telemetry in conscious rats over one week and aortic stiffness, as evidenced by increased aortic pulse wave velocity (173.2 {+/-} 50.9 mm/s vs. - 10.7 {+/-} 54.6 mm/s in controls, p = 0.0393). NEW & NOTEWORTHYWe show that renal denervation mitigates the rise in blood pressure (BP) in a model that is not genetic nor diet-dependent, the chronic mild stress model (CMS). Specifically, we demonstrate the role of afferent, rather than efferent, renal nerves in mediating the rise in BP in male CMS rats. Finally, we report that renal sympathetic nerve activity, but not BP, is elevated in female CMS rats measured by telemetry over seven days in conscious rats.

Objective: To describe cardiovascular and autonomic responses to body weight-supported treadmill training (BWSTT) combined with active or sham transcutaneous spinal cord stimulation (TSCS) in individuals with chronic, motor-complete spinal cord injury (SCI). Design and setting: Exploratory case series from randomized, sham-controlled clinical trial in a tertiary Rehabilitation Centre in Vancouver, Canada. Participants: Eight adults with chronic ([&ge;]1 year post-injury) traumatic, motor-complete (American Spinal Injury Association Impairment Scale A-B) SCI at or above T6 Interventions: Participants were randomized to 12 weeks of BWSTT plus lumbosacral TSCS or BWSTT plus sham stimulation, delivered 3 sessions/week. TSCS was delivered at T11-L1 using 30 Hz stimulation with a 10 kHz carrier frequency. Five participants completed the intervention, and four completed full cardiovascular testing (TSCS n=2; sham n=2). Outcome measures: Ambulatory blood pressure (BP) monitoring, participant-reported symptoms of AD and OH (via ADFSCI questionnaire), BP variability, orthostatic hemodynamics, echocardiography, electrocardiography (ECG)- and heart rate variability (HRV)-derived indices, and baroreflex function. Results: Among complete cases, several cardiovascular indices changed over time, including reduced daytime hypotensive burden in TSCS participants, preserved nocturnal dipping, and small changes in stroke volume and ECG-derived variability indices; however, responses were heterogeneous and overlapped with Sham. Both TSCS and Sham participants showed reduced autonomic symptom scores, while low-frequency blood pressure variability responses during orthostatic stress were heterogeneous and did not indicate a pattern that was specific to a cohort. Conclusion: Although preliminary, this exploratory complete-case analysis suggests that cardiovascular responses to BWSTT with active or sham TSCS are measurable but highly individualized after chronic motor-complete SCI. Given the small sample and overlapping Sham responses, findings are exploratory and larger trials are needed to determine whether TSCS augments cardiovascular autonomic adaptations to locomotor training.

The urinary bladder functions to store and release urine, yet how the sensation of bladder fullness is conveyed and perceived to the central nervous system is not understood. During bladder filling, the detrusor smooth muscle (DSM) generates phasic contractions, resulting in pressure fluctuations within the bladder. These transient pressure events drive bursts of afferent nerve activity, yet the underlying mechanism leading to rhythmic contractions remains unclear. Here, we examined the role of Gq protein-coupled receptor (GqPCR) activity on DSM excitability and contractility. Using ex vivo pressurized urinary bladder preparations and sharp microelectrode experiments on bladder strips from mice, we evaluated whole bladder transient pressure events, whole bladder DSM Ca2+ activity, and membrane potential in bladder strips. We found that global inhibition of urinary bladder GqPCR activity with YM-254890 abates phasic contractility and transient pressure events through a reduction in DSM Ca2+ activity and propagation of Ca2+ waves. Further, we found inhibition of GqPCR significantly hyperpolarizes DSM, reducing action potentials and decreasing excitability, and activation of protein kinase C restores membrane potential to baseline levels. These findings highlight that GqPCR activity mediates DSM excitability and contractility in such a way as to result in phasic detrusor contractions and transient pressure events.

Sex-related differences in force steadiness are often attributed to maximal strength and motor unit (MU) properties, but their independent contributions remain unclear. This study strength-matched females and males to remove the influence of maximal strength and determine whether MU properties are associated with sex-related differences in force steadiness. Twelve young adults (6 females) were matched for elbow flexion strength (females, 188.6{+/-}15.6 N; males, 199.7{+/-}24.8 N, p=0.4). Both groups performed submaximal isometric elbow flexion contractions at 2.5%, 5%, 10%, 15%, and 25% MVC. The MU recruitment thresholds (RT), discharge rates (MUDR), and coefficient of variation of interspike intervals (CVISI) were measured from intramuscular fine wire electromyography (EMG) electrodes. Force steadiness was quantified as the standard deviation (SD) and coefficient of variation (CV) of force. Across forces, SD and CV of force did not differ between females and males (p>0.05). Females had a higher recruitment threshold than males (p<0.05). Females had higher MUDR at 15% and 25% MVC (p<0.02), while males were higher at 5% MVC (p=0.02). The CVISI was greater in females (p<0.001) and positively correlated with SD of force (r=0.2) and negatively with CV of force (r=-0.2) in females and males. When strength was matched, sex-related differences in force steadiness were not evident. However, females exhibited higher MU recruitment thresholds, MUDR and CVISI. Despite greater CVISI in females, these differences did not translate into greater force fluctuations, suggesting that individual MU discharge variability is not a primary predictor of force steadiness when maximal strength is controlled. NEW & NOTEWORTHYO_LIStrength matching eliminated sex-related differences in elbow flexor force steadiness. C_LIO_LIFemales achieved similar force steadiness using higher MU recruitment thresholds and discharge rates, particularly in the short head of the biceps brachii. C_LIO_LIIn females, the greater variability in motor unit discharge was not associated with reduced force steadiness. C_LI

Objectives To validate two computational fluid dynamics (CFD) models derived from computed tomography angiography (CTA) for estimating trans-stenotic pressure gradients, using invasive intra-arterial pressure measurements as the reference standard in patients with renal artery stenosis (RAS). Background We assessed whether non-invasive assessment of the pressure gradient using CFD could be a reliable alternative to intra-arterial measurements for identifying hemodynamically significant RAS. Methods We performed intra-arterial measurements at rest and during dopamine-induced hyperemia to assess the trans-stenotic pressure gradient in 28 patients with RAS. A pre-intervention CTA scan was used to simulate the pressure gradient with a CFD model using a strategy based on Murray's law (CFD-Mu) and cortical volume (CFD-C). The agreement between the simulated and measured pressure gradients was assessed using intraclass correlation coefficients (ICC), Bland-Altman analysis and diagnostic agreement on the presence of a hemodynamically significant stenosis. Results In 20 patients, successful measurements and simulations were obtained. The ICC between measured pressure gradient and the CFD pressure gradient was 0.78 and 0.94 during baseline and 0.86 and 0.72 during hyperemia, for CFD-Mu and CFD-C, respectively. The sensitivity of CFD-Mu and CFD-C was 70% for both models at rest and 100% compared to the hyperemic measurements, whereas the specificity was 90% and 70% at rest and 79% and 72% during hyperemia, respectively. Conclusions The results support the use of individualized CFD simulations for hemodynamic assessment of RAS using CTA as input. The CFD models demonstrated high accuracy for the identification of a hemodynamically significant stenosis.

Introduction: Pediatric renal vein thrombosis is a rare but well-recognized form of venous thromboembolism. While long-term renal outcomes of neonatal cases are well-described, they are relatively unknown in cases affecting older children. Moreover, the ability of anticoagulation treatment to prevent these outcomes remains unknown. The objective of this study was to assess adverse long-term renal outcomes and determine if anticoagulation reduced their likelihood. Methods: Administrative data analysis utilizing the Pediatric Health Information System database. Renal vein thrombosis occurring in patients under 18 years were assessed. Cases involving tumor thrombus were excluded to focus the analysis only on thrombotic disease. Demographics, co-morbid conditions, anticoagulant therapies, and renal outcomes were assessed over a 9-year period. In sub-analyses, neonatal ([&le;]28 days) and non-neonatal renal vein thromboses were assessed to determine how their characteristics may differ. Results: 383 eligible renal vein thrombosis cases with 796 patient-years of follow-up were identified for analysis. 48.8% of the cases occurred in neonates. 25.3% of the cases occurred in children with pre-existing complex chronic conditions and 9.1% were associated with the onset of nephrotic syndrome. Mortality followed 15.1% of the cases, but causality cannot be assigned from administrative data. Most (80.2%) of the cases were treated with anticoagulation. Acute kidney injury occurred in 24% of cases, chronic kidney disease developed in 22.2%, hypertension in 26.9%, and proteinuria in 3.1%. Anticoagulation did not have a discernable effect on the likelihood of these long-term renal outcomes. Conclusion: Acute kidney injury, chronic kidney disease, and hypertension are prevalent in survivors of childhood renal vein thrombosis. Anticoagulation does not appear to reduce the incidence of long-term renal outcomes, but the low percentage of non-anticoagulated patients suggests treatment bias. Long-term kidney health surveillance is warranted in pediatric renal vein thrombosis survivors.

Arginine vasopressin (AVP) increases excitability of myometrial smooth muscle cells (MSMCs) through Gq-coupled AVP receptors. Although excitability requires membrane depolarization, the mechanisms linking AVP receptor activation to membrane depolarization and Ca{superscript 2} signaling are incompletely understood. Here, we show that AVPR1 is the predominant AVP receptor in primary MSMCs. In Xenopus oocytes, AVP signals through AVPR1 to inhibit SLO2.1-mediated potassium currents, reducing current amplitude to approximately 60% of control currents. Consistent with suppression of a hyperpolarizing conductance, AVP depolarized a myometrial cell line (hTERT-HM) and increased intracellular Ca{superscript 2} signaling. Analysis of Ca{superscript 2} dynamics revealed that the initial Ca{superscript 2} peak was largely preserved under conditions limiting extracellular Ca{superscript 2} entry, consistent with intracellular store release. Conversely, the oscillatory phase depended on extracellular Ca{superscript 2} influx and was reduced by SLO2.1 knockdown. Together, these findings support a model in which AVP preferentially signals through AVPR1A to inhibit SLO2.1, depolarize myometrial cells, enhance VDCC-dependent Ca{superscript 2} entry, and promote excitability, enhancing conditions for uterine contraction.

Introduction. Skeletal muscle loss in sarcopenia and neuromuscular disorders remains a major unmet medical need. AAV9-delivered follistatin (FST), a myostatin/activin antagonist, induces muscle hypertrophy; however, fibre growth without adequate vascular adaptation may limit therapeutic efficacy. We evaluated whether co-administration of a VEGF-A165 plasmid enhances the hypertrophic and angiogenic effects of intramuscular AAV-FST gene transfer in C57BL/6 mice. Methods. Thirty-six C57BL/6 mice (18 males, 18 females) were assigned to PBS vehicle (n=10), AAV-FST (1 x 10^11 vg; n=10), VEGF plasmid (100 ug; n=6), or combination treatment (VEGF plus AAV-FST; n=10). The contralateral hindlimb served as an internal control. Endpoints at Day 115 included hindlimb muscle mass ratio (R/L), transgene expression, FST protein levels, muscle fibre morphometry, capillary density, and safety assessments. Results. Combination therapy produced the highest R/L ratio (1.176 +/- 0.091; p=0.004; d=2.04), whereas AAV-FST alone showed a borderline effect (R/L=1.113; p=0.050). Compared with AAV-FST monotherapy, combination treatment increased muscle FST mRNA approximately 2.1-fold, protein levels approximately 2.0-fold, and the muscle-to-liver expression ratio 2.6-fold. It also induced larger muscle fibres and doubled CD31+ vessel counts versus AAV-FST alone, indicating simultaneous hypertrophy and angiogenesis. No adverse haematological, biochemical, or histopathological findings were observed. Discussion. Combined AAV-FST and VEGF therapy enhanced local muscle hypertrophy, increased capillary density, and improved the muscle-to-liver transgene expression profile compared with AAV-FST monotherapy. The regimen was well tolerated and supports further evaluation of angiogenic preconditioning as a strategy to improve muscle-directed gene therapy for muscle-wasting disorders.

Background: Growing evidence suggests persistent thrombotic endothelial damage (together with elevated (fibrinaloid) microclot complexes (FMCs)) and immune dysfunction in the pathophysiology of Long COVID. Recently we proposed that there are different FMC phenotypes. Here we seek to determine the nature of these FMCs and aggregates in platelet-poor plasma (PPP) by using different markers, as well as thromboelastography (TEG) to assess for hypercoagulability of samples. Material and Methods: Whole-blood and PPP from control (n=19) and Long COVID (n=20) participants were assessed by thromboelastography. FMCs were quantified by imaging flow cytometry of Thioflavin-T (ThT)-stained PPP, 10X diluted PPP, and resuspended PPP pellets. The resuspended pellets were separately stained with a CD62P-PE antibody or Hoechst 33342 to label aggregates and FMCs containing amyloid, platelet, and nuclear material. ThT and CellMask Red were co-stained for confocal microscopy. ThT and myeloperoxidase (MPO), and ThT, Congo Red, and Hoechst were co-stained for fluorescence and polarized microscopy. Whole-blood smears were imaged by scanning electron microscopy (SEM). Results: Long COVID samples showed pronounced hypercoagulability in both whole blood and PPP, with shortened R, K and TMRTG and elevated alpha-angle and MRTG, but unchanged MA and TTG, indicating altered clotting kinetics. Persistence of this phenotype in PPP implicates soluble plasma constituents. ThT-positive FMCs were significantly increased in Long COVID across undiluted, diluted, and resuspended pellet samples; counts were processing-sensitive and a substantial ThT-positive population remained in the supernatant after centrifugation, indicating heterogeneity in density. Across probes, leukocyte material was the most abundant, then platelet material, and ThT-positive FMCs were the least abundant, with the three populations exhibiting unique morphology and occupying distinct size domains. Platelet-derived material was significantly elevated in Long COVID, whereas nuclear material was not. Co-stained samples subject to confocal, fluorescence, and polarized microscopy imaging showed that FMCs are heterogeneous, including events positive for ThT, CellMask, Hoechst, MPO, and Congo Red, and also a distinct subset of membrane-free, ThT-only events. Conclusion: In this Long COVID cohort, plasma is characterised by hypercoagulability and an increased burden of ThT-positive FMCs that are numerically minor relative to, and morphologically distinct from, aggregates and amyloidogenic FMCs marked with platelet- and leukocyte-derived material. The increased burden of platelet debris in PPP is likely indicative of persistent platelet activity. The existence of membrane-free, ThT-only FMCs, in addition to FMCs associated with cellular material, confirms an amyloid-dominated FMC population. Furthermore, positive Congo Red signal further confirms the amyloid nature of FMCs in PPP.

Spinal cord injury (SCI) disrupts innervation to the lower urinary tract, resulting in bladder dysfunction that predisposes to urinary infections and renal impairment. While inflammation is central to bladder pathology after SCI, the molecular events linking acute to chronic remodeling are poorly defined. We hypothesized that early treatment with pirfenidone, an anti-inflammatory and anti-fibrotic drug, would attenuate bladder pathology after SCI. Adult female C57BL/6J mice underwent contusive SCI or sham laminectomy, and bladders were collected at 2, 7, 16, and 45 days later. SCI induced bladder hypertrophy, edema, hemorrhage, neutrophil infiltration, cell proliferation and loss of voiding function in the first 48 hours. Transcriptomic profiling at this timepoint was characterized by activation of inflammatory and cytokine pathways including TNFalpha, IL-6, the complement cascade, and TGFbeta. Although bladder function partially recovered by day 7, inflammatory pathways persisted and extracellular matrix (ECM) remodeling programs emerged. By day 16, robust activation of ECM-remodeling pathways was evident in all bladders. Treatment with pirfenidone during the acute inflammatory phase (day 2-7) reduced bladder hypertrophy and suppressed expression of pro-fibrotic, inflammatory, and neuroplasticity-associated genes including Bdnf and Chrm2 that encodes muscarinic receptor 2 (M2). Mechanistically, pirfenidone attenuated TGFbeta signaling as shown by downregulation of phosphoSmad2 protein in whole bladders and decreased M2 receptor expression in the urothelium. These molecular changes correlated with improved function in pirfenidone-treated mice as shown by fewer voiding events with larger urine volumes up until 45 days after SCI. Early treatment with pirfenidone limits inflammation and fibrosis, normalizes neural signaling, and improves bladder function after SCI.

The Notch signaling pathway plays a central role in development and cell fate determination. Its function depends on tightly regulated intracellular trafficking of the Notch receptor and the Notch intracellular domain (NICD) after cleavage by {gamma}-secretase. Notch signaling is essential for principal cell differentiation within the renal collecting duct and for proximal-distal patterning during kidney development. Notch activity has also been shown to influence the trafficking of several membrane proteins, including nephrin in kidney cells and monocarboxylate transporter 1 in brain endothelial cells. Aquaporin-2 (AQP2) is the key vasopressin-regulated water channel in the collecting duct, and proper AQP2 trafficking and recycling are required for physiologically appropriate urine concentration. To determine whether and, if so, how Notch signaling modulates AQP2 trafficking, we performed studies using LLCPK1 renal epithelial cells stably expressing AQP2 (LLCPK1-AQP2). Exposing cells to 35 M DAPT (which inhibits y-secretase, preventing cleavage and activation of Notch receptor signaling) for 30 min significantly increased AQP2 membrane accumulation in LLCPK1-AQP2 cells as revealed by immunofluorescence staining. Using a rhodamine-transferrin internalization assay, we found that DAPT reduced clathrin-mediated endocytosis by 60%. This blockade increases AQP2 membrane accumulation by preventing the reinternalization of AQP2 that is delivered to the plasma membrane by exocytosis during its constitutive recycling pathway. Using an F-actin polymerization assay, we then found that Notch inhibition decreases F-actin polymerization by de-activating the small GTPase RhoA, using GSTRBD, a substrate that binds to active RhoA, as seen by western blotting using phospho-specific antibodies. Because actin polymerization is required for AQP2 endocytosis, RhoA inhibition by DAPT would result in the decreased internalization of AQP2 that we observed by immunofluorescence. While the mechanism by which DAPT inhibits RhoA activity remains to be determined, our study shows that AQP2 trafficking is regulated by the Notch signaling pathway in vitro and suggests that modulation of Notch signaling may represent a novel strategy to address water balance disorders that involve defects in the AQP2 trafficking process.

Background: The dietary approaches to stop hypertension (DASH) diet is often recommended to patients with chronic kidney disease, although evidence regarding its efficacy in this population is limited. Our study tested the hypothesis that increased adherence to the dietary approaches to stop hypertension (DASH) diet score would be associated with longer time to kidney function decline among Veterans. Methods: We conducted a retrospective cohort study of 251,921 Veterans enrolled in the Million Veteran Program (MVP). The DASH diet score was calculated from the food frequency questionnaire and categorized into tertiles. The primary outcome was a composite of: Kidney event or death, where a kidney event was defined as a sustained 40% decline in estimated glomerular filtration rate (eGFR) or end-stage kidney disease (ESKD). Cox regression models compared the hazard for both outcomes by DASH score tertiles. We examined modification by ancestry, sex and other clinical characteristics Results: The median age was 67 years and 90% of Veterans were men. There were 59,269 (23.5%) who experienced the primary composite outcome, during the maximum follow-up of 10 years (median 6.1 years). Crude incidence rates for the kidney event and death outcome were 43.3, 40.4, and 36.8 per 1000 person-years of DASH score by tertiles. DASH score was associated with a lower hazard ratio (HR) for the primary composite outcome; third vs first tertile 0.81 (95% Confidence Interval (CI) 0.80 - 0.83) and second vs first tertile HR 0.90 [95% CI 0.88 - 0.92]. In subgroup analysis for individuals of African ancestry, Admixed American, and Females, only the third tertile of the DASH score was associated with a statistically significant reduction in composite outcome. Conclusion: Beneficial associations of the DASH diet were observed across subgroups. Future research is needed to understand gene and environmental factors that influence the observed subgroup differences