Biallelic USP14 variants cause a syndromic neurodevelopmental disorder.

PURPOSE: Imbalances in protein homeostasis affect human brain development, with the ubiquitin-proteasome system (UPS) and autophagy playing crucial roles in neurodevelopmental disorders (NDD). This study explores the impact of biallelic USP14 variants on neurodevelopment, focusing on its role as a key hub connecting UPS and autophagy. METHODS: Here, we identified biallelic USP14 variants in 4 individuals from 3 unrelated families: 1 fetus, a newborn with a syndromic NDD and 2 siblings affected by a progressive neurological disease. Specifically, the 2 siblings from the latter family carried 2 compound heterozygous variants c.8T>C p.(Leu3Pro) and c.988C>T p.(Arg330∗), whereas the fetus had a homozygous frameshift c.899_902del p.(Lys300Serfs∗24) variant, and the newborn patient harbored a homozygous frameshift c.233_236del p.(Leu78Glnfs∗11) variant. Functional studies were conducted using sodium dodecyl-sulfate polyacrylamide gel electrophoresis, western blotting, and mass spectrometry analyses in both patient-derived and CRISPR-Cas9-generated cells. RESULTS: Our investigations indicated that the USP14 variants correlated with reduced N-terminal methionine excision, along with profound alterations in proteasome, autophagy, and mitophagy activities. CONCLUSION: Biallelic USP14 variants in NDD patients perturbed protein degradation pathways, potentially contributing to disorder etiology. Altered UPS, autophagy, and mitophagy activities underscore the intricate interplay, elucidating their significance in maintaining proper protein homeostasis during brain development.

Loss of Cardiac Splicing Regulator RBM20 Is Associated With Early-Onset Atrial Fibrillation.

We showed an association between atrial fibrillation and rare loss-of-function (LOF) variants in the cardiac splicing regulator RBM20 in 2 independent cohorts. In a rat model with loss of RBM20, we demonstrated altered splicing of sarcomere genes (NEXN, TTN, TPM1, MYOM1, and LDB3), and differential expression in key cardiac genes. We identified altered sarcomere and mitochondrial structure on electron microscopy imaging and found compromised mitochondrial function. Finally, we demonstrated that 3 novel LOF variants in RBM20, identified in patients with atrial fibrillation, lead to significantly reduced splicing activity. Our results implicate alternative splicing as a novel proarrhythmic mechanism in the atria.

Unveiling the crucial neuronal role of the proteasomal ATPase subunit gene PSMC5 in neurodevelopmental proteasomopathies.

Neurodevelopmental proteasomopathies represent a distinctive category of neurodevelopmental disorders (NDD) characterized by genetic variations within the 26S proteasome, a protein complex governing eukaryotic cellular protein homeostasis. In our comprehensive study, we identified 23 unique variants in PSMC5 , which encodes the AAA-ATPase proteasome subunit PSMC5/Rpt6, causing syndromic NDD in 38 unrelated individuals. Overexpression of PSMC5 variants altered human hippocampal neuron morphology, while PSMC5 knockdown led to impaired reversal learning in flies and loss of excitatory synapses in rat hippocampal neurons. PSMC5 loss-of-function resulted in abnormal protein aggregation, profoundly impacting innate immune signaling, mitophagy rates, and lipid metabolism in affected individuals. Importantly, targeting key components of the integrated stress response, such as PKR and GCN2 kinases, ameliorated immune dysregulations in cells from affected individuals. These findings significantly advance our understanding of the molecular mechanisms underlying neurodevelopmental proteasomopathies, provide links to research in neurodegenerative diseases, and open up potential therapeutic avenues.

Identification of eight novel proteasome variants in five unrelated cases of proteasome-associated autoinflammatory syndromes (PRAAS).

Mutations in genes coding for proteasome subunits and/or proteasome assembly helpers typically cause recurring autoinflammation referred to as chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperatures (CANDLE) or proteasome-associated autoinflammatory syndrome (PRAAS). Patients with CANDLE/PRAAS present with mostly chronically elevated type I interferon scores that emerge as a consequence of increased proteotoxic stress by mechanisms that are not fully understood. Here, we report on five unrelated patients with CANDLE/PRAAS carrying novel inherited proteasome missense and/or nonsense variants. Four patients were compound heterozygous for novel pathogenic variants in the known CANDLE/PRAAS associated genes, PSMB8 and PSMB10, whereas one patient showed additive loss-of-function mutations in PSMB8. Variants in two previously not associated proteasome genes, PSMA5 and PSMC5, were found in a patient who also carried the PSMB8 founder mutation, p.T75M. All newly identified mutations substantially impact the steady-state expression of the affected proteasome subunits and/or their incorporation into mature 26S proteasomes. Our observations expand the spectrum of PRAAS-associated genetic variants and improve a molecular diagnosis and genetic counseling of patients with sterile autoinflammation.

Establishing 20S Proteasome Genetic, Translational and Post-Translational Status from Precious Biological and Patient Samples with Top-Down MS.

The mammalian 20S catalytic core of the proteasome is made of 14 different subunits (α1-7 and ß1-7) but exists as different subtypes depending on the cell type. In immune cells, for instance, constitutive catalytic proteasome subunits can be replaced by the so-called immuno-catalytic subunits, giving rise to the immunoproteasome. Proteasome activity is also altered by post-translational modifications (PTMs) and by genetic variants. Immunochemical methods are commonly used to investigate these PTMs whereby protein-tagging is necessary to monitor their effect on 20S assembly. Here, we present a new miniaturized workflow combining top-down and bottom-up mass spectrometry of immunopurified 20S proteasomes that analyze the proteasome assembly status as well as the full proteoform footprint, revealing PTMs, mutations, single nucleotide polymorphisms (SNPs) and induction of immune-subunits in different biological samples, including organoids, biopsies and B-lymphoblastoid cell lines derived from patients with proteasome-associated autoinflammatory syndromes (PRAAS). We emphasize the benefits of using top-down mass spectrometry in preserving the endogenous conformation of protein modifications, while enabling a rapid turnaround (1 h run) and ensuring high sensitivity (1-2 pmol) and demonstrate its capacity to semi-quantify constitutive and immune proteasome subunits.

Colchicine enhances ß adrenoceptor-mediated vasodilation in men with essential hypertension.

AIMS: The aim of this study is to examine whether colchicine improves ß adrenoceptor-mediated vasodilation in humans by conducting a double-blinded, placebo-controlled intervention study. Colchicine treatment has known beneficial effects on cardiovascular health and reduces the incidence of cardiovascular disease. Studies in isolated rodent arteries have shown that colchicine can enhance ß adrenoceptor-mediated vasodilation, but this has not been determined in humans. METHODS: Middle-aged men with essential hypertension were randomly assigned firstly to acute treatment with either 0.5 mg colchicine (n = 19) or placebo (n = 12). They were subsequently re-randomized for 3 weeks of treatment with either colchicine 0.5 mg twice daily (n = 16) or placebo (n = 15) followed by a washout period of 48-72 h. The vasodilator responses to isoprenaline, acetylcholine and sodium nitroprusside were determined as well as arterial pressure, arterial compliance and plasma inflammatory markers. RESULTS: Acute colchicine treatment increased isoprenaline (by 38% for the highest dose) as well as sodium nitroprusside (by 29% main effect) -induced vasodilation but had no effect on the response to acetylcholine. The 3-week colchicine treatment followed by a washout period did not induce an accumulated or sustained effect on the ß adrenoceptor response, and there was no effect on arterial pressure, arterial compliance or the level of measured inflammatory markers. CONCLUSION: Colchicine acutely enhances ß adrenoceptor- and nitric oxide-mediated changes in vascular conductance in humans, supporting that the mechanism previously demonstrated in rodents, translates to humans. The results provide novel translational evidence for a transient enhancing effect of colchicine on ß adrenoceptor-mediated vasodilation in humans with essential hypertension. CONDENSED ABSTRACT: Preclinical studies in isolated rodent arteries have shown that colchicine can enhance ß adrenoceptor-mediated vasodilation. Here we show that this effect of colchicine can be translated to humans. Acute colchicine treatment was found to increase both isoprenaline- and sodium nitroprusside-induced vasodilation. The study provides the first translational evidence for a transient ß adrenoceptor-mediated vasodilatory effect of colchicine in humans. The finding of an acute effect suggests that it may be clinically important to maintain an adequate bioavailability of colchicine.

Dupilumab Demonstrates Rapid Onset of Action in Improving Signs, Symptoms and Quality of Life in Adults with Atopic Dermatitis.

INTRODUCTION: Dupilumab has significantly improved the signs, symptoms and quality of life (QoL) of patients with moderate-to-severe atopic dermatitis (AD) in randomised, controlled clinical trials. However, there is a need to assess the effectiveness and safety of dupilumab in real-world clinical practice. The PROLEAD study was designed to examine the effectiveness and safety of dupilumab in moderate-to-severe AD in a real-world setting in Germany. Here, we present 12-week effectiveness and safety results with dupilumab from PROLEAD. METHODS: PROLEAD is a multicentre, prospective, non-interventional study being conducted at 126 routine care sites across Germany. Adults with moderate-to-severe AD who require systemic therapy were treated with dupilumab as indicated by the Summary of Product Characteristics. Data collected included physician assessments (EASI, BSA, SCORAD, and IGA) and patient-reported outcomes (PROs [POEM, DLQI, EQ-5D-5L, Peak Pruritus NRS and MOS Sleep Scale]). RESULTS: Of 839 patients assessed for eligibility, 828 were included. The full analysis and safety analysis sets comprised 775 and 818 patients, respectively. The number of patients receiving concomitant therapy decreased from baseline to Week 12. Mean (standard deviation [SD]) percentage change in EASI score from baseline to Week 12 was -67.5% (48.4%) and was comparable across the four body regions. The proportion of patients achieving EASI-75 was 59.4% at Week 12. Mean (SD) Peak Pruritus NRS decreased from 7.4 (2.3) at baseline to 3.4 (2.6) at Week 12. Improvements from baseline to Week 12 were reported in all PROs assessed. No new safety signals were observed. DISCUSSION: Improvements in efficacy outcomes and adverse event rates in a real-world setting were more favourable than in phase 3 clinical trials. CONCLUSIONS: The 12-week findings of PROLEAD demonstrate that treatment with dupilumab is effective and well tolerated, with rapid onset of action in signs, symptoms and QoL in patients with moderate-to-severe AD in the real world. TRIAL REGISTRATION NUMBER: DUPILL08907; NIS-Nr. 433.

Sympathetic activity is not a main cause of blood pressure reduction with exercise training in un-medicated middle-aged/older men.

BACKGROUND: This study tested the hypothesis that training reduces resting sympathetic activity and improves baroreflex control in both hypertensive and normotensive men but reduces blood pressure only in hypertensive men. METHODS: Middle-aged/older un-medicated stage-1 hypertensive males (mean age 55 ± 3 years; n = 13) and normotensive controls (mean age 60 ± 5 years; n = 12) participated in 8 weeks of supervised high-intensity interval spinning training. Before and after training, muscle sympathetic nerve activity (MSNA) and blood pressure were measured at rest and during a sympatho-excitatory cold pressor test (CPT). Based on the measurements, baroreceptor sensitivity and baroreceptor threshold were calculated. RESULTS: Resting MSNA and baroreceptor sensitivity were similar for the hypertensive and the normotensive groups. Training lowered MSNA (p < 0.05), expressed as burst frequency (burst/min), overall, and to a similar extent, in both groups (17% and 27%, respectively, in hypertensive and normotensive group), whereas blood pressure was only significantly (p < 0.05) lowered (by 4 mmHg in both systolic and diastolic pressure) in the hypertensive group. Training did not (p > 0.05) alter the MSNA or blood pressure response to CPT or increase baroreceptor sensitivity but reduced (p < 0.05) the baroreceptor threshold with a main effect for both groups. Training adherence and intensity were similar in both groups yet absolute maximal oxygen uptake increased by 15% in the normotensive group only. CONCLUSION: The dissociation between the training induced changes in resting MSNA, lack of change in baroreflex sensitivity and the change in blood pressure, suggests that MSNA is not a main cause of the blood pressure reduction with exercise training in un-medicated middle-aged/older men.

Dupilumab Treatment of Atopic Dermatitis in Routine Clinical Care: Baseline Characteristics of Patients in the PROLEAD Prospective, Observational Study.

INTRODUCTION: Dupilumab is the first biologic licensed to treat patients with moderate-to-severe atopic dermatitis (AD) who require systemic therapy. PROLEAD was designed to document the real-world effectiveness and safety of dupilumab in patients with moderate-to-severe AD. The present study aims to describe the baseline characteristics of patients treated with dupilumab in Germany. METHODS: PROLEAD is a national, multicentre, prospective, non-interventional study, with a 2-year observation period. Adults with moderate-to-severe AD treated with dupilumab were included. Baseline characteristics, physician assessments, and patient-reported outcomes (PROs) were collected. RESULTS: The study involved 126 sites throughout Germany. Of 839 patients assessed for eligibility, 828 were included, with baseline data available for 817 patients. Mean (standard deviation, SD) age of patients was 43.4 (15.8) years, with 396 (48.5%) patients being female. Overall, 66.6% of patients received their first diagnosis of AD during childhood. In total, 423 (51.8%) patients had co-existing atopic and type 2 inflammatory diseases, including allergic conjunctivitis (36.8%) and bronchial asthma (22.5%). Overall, 61.4% of patients had received systemic therapy, most commonly oral corticosteroids (49.9%). Approximately half of patients (51.3%) had received UV/phototherapy prior to baseline. Treatment with moderate-potent (Class 2) or potent (Class 3) topical corticosteroids was the most common concomitant treatment at baseline. However, 50.4% of patients had not received concomitant AD treatment with dupilumab at baseline. The most reported reason for initiating dupilumab was "Topical therapy alone was not sufficient" (95.1%). Mean (SD) physician assessments: EASI: 22.9 (14.5); SCORAD: 63.3 (16.2); IGA: 3.3 (0.7). Mean (SD) PROs: DLQI: 13.9 (7.1); peak pruritus NRS: 7.4 (2.3). CONCLUSIONS: Patients with moderate-to-severe AD present a long medical history, impaired quality of life, and high prevalence of co-existing type 2 inflammatory diseases. Dupilumab was used as a first-line systemic treatment in 38.6% of patients.

Exercise Training Lowers Arterial Blood Pressure Independently of Pannexin 1 in Men with Essential Hypertension.

INTRODUCTION: Regular exercise training reduces arterial blood pressure, but the underlying mechanisms are unclear. Here, we evaluated the potential involvement of pannexin 1, an ATP releasing channel, in the blood pressure-reducing effect of training. METHODS: Middle-age men, 13 normotensive and 14 nonmedicated stage 1 hypertensive, completed 8 wk of intensive aerobic cycle training. Before and after training, blood pressure and changes in leg vascular conductance, induced by femoral arterial infusion of tyramine (induces endogenous noradrenaline release), acetylcholine, or sodium nitroprusside, were measured during control conditions and after acute pannexin 1 inhibition by probenecid. A skeletal muscle biopsy was obtained from the thigh, pre- and posttraining. RESULTS: Exercise training reduced mean systolic and diastolic blood pressure by ~5 ( P = 0.013) and 5 mm Hg ( P < 0.001), respectively, in the hypertensive group only. The reduction in blood pressure was not related to changes in pannexin 1 function because mean arterial blood pressure and tyramine-induced vasoconstriction remain unaltered by pannexin 1 inhibition after training in both groups. After training, pannexin 1 inhibition enhanced leg vascular conductance in the normo- and hypertensive groups at baseline (41.5%, P = 0.0036, and 37.7%, P = 0.024, respectively) and in response to sodium nitroprusside infusion (275%, P = 0.038, and 188%, P = 0.038, respectively). Training did not alter the pannexin 1 protein expression in skeletal muscle. Training enhanced the vasodilator response to acetylcholine infusion and increased the expression of microvascular function-relevant proteins. CONCLUSIONS: The exercise training-induced lowering of arterial blood pressure in nonmedicated hypertensive men does not involve an altered function of pannexin 1.

Is the Pannexin-1 Channel a Mechanism Underlying Hypertension in Humans? a Translational Study of Human Hypertension.

BACKGROUND: In preclinical models, the pannexin-1 channel has been shown to be involved in blood pressure regulation through an effect on peripheral vascular resistance. Pannexin-1 releases ATP, which can activate constrictive purinergic receptors on the smooth muscle cells. Pannexin-1 opening is proposed to be mediated by α-adrenergic receptors to potentiate sympathetic constriction. This positions pannexin-1 as a putative pharmacological target in blood pressure regulation in humans. The aim was to provide the first translational evidence for a role of pannexin-1 in essential hypertension in humans by use of an advanced invasive mechanistic approach. METHODS: Middle-aged stage-1 hypertensive (n=13; 135.7±6.4 over 83.7±3.7 mm Hg) and normotensive men (n=12; 117.3±5.7 over 72.2±3.5 mm Hg) were included. Blood pressure and leg vascular resistance were determined during femoral arterial infusion of tyramine (α-adrenergic receptor stimulation), sodium nitroprusside, and acetylcholine. Measurements were made during control conditions and with pannexin-1 blockade (3000 mg probenecid). Expression of Pannexin-1, purinergic- and α-adrenergic receptors in skeletal muscle biopsies was determined by Western blot. RESULTS: The changes in leg vascular resistance in response to tyramine (+289% versus +222%), sodium nitroprusside (-82% versus -78%) and acetylcholine (-40% versus -44%) infusion were not different between the 2 groups (P>0.05) and pannexin-1 blockade did not alter these variables (P>0.05). Expression of pannexin-1 and of purinergic- and α-adrenergic receptors was not different between the 2 groups (P>0.05). CONCLUSIONS: Contrary to our hypothesis, the data demonstrate that pannexin-1 does not contribute to the elevated blood pressure in essential hypertension, a finding, which also opposes that reported in preclinical models.

Redox balance in human skeletal muscle-derived endothelial cells - Effect of exercise training.

Aerobic training can improve vascular endothelial function in-vivo. The aim of this study was to elucidate the mechanisms underlying this improvement in isolated human microvascular endothelial cells. Sedentary males, aged 57 ± 6 years completed 8 weeks of intense aerobic training. Resting muscle biopsies were obtained from the thigh muscle and used for isolation of endothelial cells (pre n = 23, post n = 16). The cells were analyzed for mitochondrial respiration, H2O2 emission, glycolysis, protein levels of antioxidants, NADPH oxidase, endothelial nitric oxide (NO) synthase and prostacyclin synthase (PGI2S). In-vivo microvascular function, assessed by acetylcholine infusion and arterial blood pressure were also determined. Endothelial mitochondrial respiration and H2O2 formation were similar before and after training whereas the expression of superoxide dismutase and the expression of glutathione peroxidase were 2.4-fold (p = 0.012) and 2.3-fold (p = 0.006) higher, respectively, after training. In-vivo microvascular function was increased by 1.4-fold (p = 0.036) in parallel with a 2.1-fold increase in endothelial PGI2S expression (p = 0.041). Endothelial cell glycolysis was reduced after training, as indicated by a 65% lower basal production of lactate (p = 0.003) and a 30% lower expression of phosphofructokinase (p = 0.011). Subdivision of the participants according to blood pressure at base-line (n = 23), revealed a 2-fold higher (p = 0.049) rate of H2O2 production in endothelial cells from hypertensive participants. Our data show that exercise training increases skeletal muscle microvascular endothelial cell metabolism, antioxidant capacity and the capacity to form prostacyclin. Moreover, elevated blood pressure is associated with increased endothelial mitochondrial ROS formation.

Functional sympatholysis in mouse skeletal muscle involves sarcoplasmic reticulum swelling in arterial smooth muscle cells.

The vasoconstrictive effect of sympathetic activity is attenuated in contracting skeletal muscle (functional sympatholysis), allowing increased blood supply to the working muscle but the underlying mechanisms are incompletely understood. The purpose of this study was to examine α-adrenergic receptor responsiveness in isolated artery segments from non-exercised and exercised mice, using wire myography. Isometric tension recordings performed on femoral artery segments from exercised mice showed decreased α-adrenergic receptor responsiveness compared to non-exercised mice (logEC50 -5.2 ± 0.04 M vs. -5.7 ± 0.08 M, respectively). In contrast, mesenteric artery segments from exercised mice displayed similar α-adrenergic receptor responses compared to non-exercised mice. Responses to the vasoconstrictor serotonin (5-HT) and vasodilator isoprenaline, were similar in femoral artery segments from non-exercised and exercised mice. To study sarcoplasmic reticulum (SR) function, we examined arterial contractions induced by caffeine, which depletes SR Ca2+ and thapsigargin, which inhibits SR Ca2+ -ATPase (SERCA) and SR Ca2+ uptake. Arterial contractions to both caffeine and thapsigargin were increased in femoral artery segment from exercised compared to non-exercised mice. Furthermore, 3D electron microscopy imaging of the arterial wall showed SR volume/length ratio increased 157% in smooth muscle cells of the femoral artery from the exercised mice, whereas there was no difference in SR volume/length ratio in mesenteric artery segments. These results show that in arteries surrounding exercising muscle, the α-adrenergic receptor constrictions are blunted, which can be attributed to swollen smooth muscle cell SR's, likely due to increased Ca2+ content that is possibly reducing free intracellular Ca2+ available for contraction. Overall, this study uncovers a previously unknown mechanism underlying functional sympatholysis.

Interaction of fibrinogen-magnetic nanoparticle bioconjugates with integrin reconstituted into artificial membranes.

Magnetic nanoparticles have a broad spectrum of biomedical applications including cell separation, diagnostics and therapy. One key issue is little explored: how do the engineered nanoparticles interact with blood components after injection? The formation of bioconjugates in the bloodstream and subsequent reactions are potentially toxic due to the ability to induce an immune response. The understanding of the underlying processes is of major relevance to design not only efficient, but also safe nanoparticles for e.g. targeted drug delivery applications. In this study, we report on maghemite nanoparticles functionalized with citrate-, dextran- and polyethylene glycol coatings and their interaction with the clotting protein fibrinogen. Further, we investigate using biophysical tools (e.g. dynamic light scattering, circular dichroism spectroscopy and quartz crystal microbalance) the interaction of the magnetic nanoparticles-fibrinogen bioconjugates with artificial cell membranes as a model system for blood platelets. We found that fibrinogen corona formation provides colloidal stability to maghemite nanoparticles. In addition, bioconjugates of fibrinogen with dextran- and citrate-coated NPs interact with integrin-containing lipid bilayer, especially upon treatment with divalent ions, whereas PEG-coating reveals minor interaction. Our study at the interface of protein-conjugated nanoparticles and artificial cell membranes is essential for engineering safe nanoparticles for drug delivery applications.

Lack of Connexins 40 and 45 Reduces Local and Conducted Vasoconstrictor Responses in the Murine Afferent Arterioles.

The juxtaglomerular apparatus (JGA) is an essential structure in the regulation of renal function. The JGA embodies two major functions: tubuloglomerular feedback (TGF) and renin secretion. TGF is one of the mechanisms mediating renal autoregulation. It is initiated by an increase in tubular NaCl concentration at the macula densa cells. This induces a local afferent arteriolar vasoconstriction and a conducted response that can be measured several 100 µm upstream from the juxtaglomerular segment. This spread of the vasomotor response into the surrounding vasculature likely plays a key role in renal autoregulation, and it requires the presence of gap junctions, intercellular pores based on connexin (Cx) proteins. Several Cx isoforms are expressed in the JGA and in the arteriolar wall. Disruption of this communication pathway is associated with reduced TGF, dysregulation of renin secretion, and hypertension. We examine if the absence of Cx40 or Cx45, expressed in the endothelial and vascular smooth muscle cells respectively, attenuates afferent arteriolar local and conducted vasoconstriction. Afferent arterioles from wildtype and Cx-deficient mice (Cx40 and Cx45) were studied using the isolated perfused juxtamedullary nephron preparation. Vasoconstriction was induced via electrical pulse stimulation at the glomerular entrance. Inner afferent arteriolar diameter was measured locally and upstream to evaluate conducted vasoconstriction. Electrical stimulation induced local vasoconstriction in all groups. The local vasoconstriction was significantly smaller when Cx40 was absent. The vasoconstriction decreased in magnitude with increasing distance from the stimulation site. In both Cx40 and Cx45 deficient mice, the vasoconstriction conducted a shorter distance along the vessel compared to wild-type mice. In Cx40 deficient arterioles, this may be caused by a smaller local vasoconstriction. Collectively, these findings imply that Cx40 and Cx45 are central for normal vascular reactivity and, therefore, likely play a key role in TGF-induced regulation of afferent arteriolar resistance.

GLP-1-induced renal vasodilation in rodents depends exclusively on the known GLP-1 receptor and is lost in prehypertensive rats.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone known to stimulate postprandial insulin release. However, GLP-1 also exerts extrapancreatic effects, including renal effects. Some of these renal effects are attenuated in hypertensive rats, where renal expression of GLP-1 receptors is reduced. Here, we assessed the expression and vascular function of GLP-1 receptors in kidneys from young prehypertensive rats. We also examined GLP-1-induced vasodilation in the renal vasculature in wild-type (WT) and GLP-1 receptor knockout mice using wire and pressure myography and the isolated perfused juxtamedullary nephron preparation. We investigated whether GLP-1 and the metabolite GLP-1(9-36)amide had renal vascular effects independent of the known GLP-1 receptor. We hypothesized that hypertension decreased expression of renal GLP-1 receptors. We also hypothesized that GLP-1-induced renal vasodilatation depended on expression of the known GLP-1 receptor. In contrast to normotensive rats, no immunohistochemical staining or vasodilatory function of GLP-1 receptors was found in kidneys from prehypertensive rats. In WT mice, GLP-1 induced renal vasodilation and reduced the renal autoregulatory response. The GLP-1 receptor antagonist exendin 9-39 inhibited relaxation, and GLP-1(9-36)amide had no vasodilatory effect. In GLP-1 receptor knockout mice, no relaxation induced by GLP-1 or GLP-1(9-36)amide was found, the autoregulatory response in afferent arterioles was normal, and no GLP-1-induced reduction of autoregulation was found. We conclude that in prehypertensive kidneys, expression and function of GLP-1 receptors is lost. The renal vasodilatory effect of GLP-1 is mediated exclusively by the known GLP-1 receptor. GLP-1(9-36)amide has no renal vasodilatory effect. GLP-1 attenuates renal autoregulation by reducing the myogenic response.

Influence of connexin45 on renal autoregulation.

Renal autoregulation is mediated by the myogenic response and tubuloglomerular feedback (TGF) working in concert to maintain renal blood flow and glomerular filtration rate despite fluctuations in renal perfusion pressure. Intercellular communication through gap junctions may play a role in renal autoregulation. We examine if one of the building blocks in gap junctions, connexin45 (Cx45), which is expressed in vascular smooth muscle cells, has an influence on renal autoregulatory efficiency. The isolated perfused juxtamedullary nephron preparation was used to measure afferent arteriolar diameter changes in response to acute changes in renal perfusion pressure. In segmental arteries, pressure myography was used to study diameter changes in response to pressure changes. Wire myography was used to study vasoconstrictor and vasodilator responses. A mathematical model of the vascular wall was applied to interpret experimental data. We found a significant reduction in the afferent arteriolar constriction in response to acute pressure increases in Cx45 knockout (KO) mice compared with wild-type (WT) mice. Abolition of TGF caused a parallel upward shift in the autoregulation curve of WT animals but had no effect in KO animals, which is compatible with TGF providing a basal tonic contribution in afferent arterioles whereas Cx45 KO animals were functionally papillectomized. Analysis showed a shift toward lower stress sensitivity in afferent arterioles from Cx45 KO animals, indicating that the absence of Cx45 may also affect myogenic properties. Finally, loss of Cx45 in vascular smooth muscle cells appeared to associate with a change in both structure and passive properties of the vascular wall.