SGLT2 inhibition promotes glomerular repopulation by cells of renin lineage in experimental kidney disease.

AIM: Sodium glucose co-transporter-2 (SGLT2) inhibitors stimulate renal excretion of sodium and glucose and exert renal protective effects in patients with (non-)diabetic chronic kidney disease (CKD) and may as well protect against acute kidney injury (AKI). The mechanism behind this kidney protective effect remains unclear. Juxtaglomerular cells of renin lineage (CoRL) have been demonstrated to function as progenitors for multiple adult glomerular cell types in kidney disease. This study assesses the impact of SGLT2 inhibition on the repopulation of glomerular cells by CoRL and examines their phenotypic commitment. METHODS: Experiments were performed in Ren1cre-tdTomato lineage-trace mice. Either 5/6 nephrectomy (5/6NX) modeling CKD or bilateral ischaemia reperfusion injury (bIRI) mimicking AKI was applied, while the SGLT2 inhibitor empagliflozin (10 mg/kg) was administered daily via oral gavage for 14 days. RESULTS: Both 5/6NX and bIRI-induced kidney injury increased the number of glomerular CoRL-derived cells. SGLT2 inhibition improved kidney function after 5/6NX, indicated by decreased blood creatinine and urea levels, but not after bIRI. In line with this, empagliflozin in 5/6NX animals resulted in less glomerulosclerosis, while it did not affect histopathological features in bIRI. Treatment with empagliflozin resulted in an increase in the number of CoRL-derived glomerular cells in both 5/6NX and bIRI conditions. Interestingly, SGLT2 inhibition led to more CoRL-derived podocytes in 5/6NX animals, whereas empagliflozin-treated bIRI mice presented with increased levels of parietal epithelial and mesangial cells derived from CoRL. CONCLUSION: We conclude that SGLT2 inhibition by empagliflozin promotes CoRL-mediated glomerular repopulation with selective CoRL-derived cell types depending on the type of experimental kidney injury. These findings suggest a previously unidentified mechanism that could contribute to the renoprotective effect of SGLT2 inhibitors.

Olodaterol exerts anti-inflammatory effects on COPD airway epithelial cells.

BACKGROUND: Airway inflammation is a key feature of chronic obstructive pulmonary disease (COPD) and inhaled corticosteroids (ICS) remain the main treatment for airway inflammation. Studies have noted the increased efficacy of ICS and long-acting beta 2 agonist (LABA) combination therapy in controlling exacerbations and improving airway inflammation than either monotherapy. Further studies have suggested that LABAs may have inherent anti-inflammatory potential, but this has not been well-studied. OBJECTIVE: We hypothesize that the LABA olodaterol can inhibit airway inflammation resulting from exposure to respiratory syncytial virus (RSV) via its binding receptor, the ß2-adrenergic receptor. METHODS: Human bronchial epithelial brushing from patients with and without COPD were cultured into air-liquid interface (ALI) cultures and treated with or without olodaterol and RSV infection to examine the effect on markers of inflammation including interleukin-8 (IL-8) and mucus secretion. The cell line NCI-H292 was utilized for gene silencing of the ß2-adrenergic receptor via siRNA as well as receptor blocking via ICI 118,551 and butaxamine. RESULTS: At baseline, COPD-ALIs produced greater amounts of IL-8 than control ALIs. Olodaterol reduced RSV-mediated IL-8 secretion in both COPD and control ALIs and also significantly reduced Muc5AC staining in COPD-ALIs infected with RSV. A non-significant reduction was seen in control ALIs. Gene silencing of the ß2-adrenergic receptor in NCI-H292 negated the ability of olodaterol to inhibit IL-8 secretion from both RSV infection and lipopolysaccharide stimulus, as did blocking of the receptor with ICI 118,551 and butaxamine. CONCLUSIONS: Olodaterol exhibits inherent anti-inflammatory properties on the airway epithelium, in addition to its bronchodilation properties, that is mediated through the ß2-adrenergic receptor and independent of ICS usage.

Second M3 muscarinic receptor binding site contributes to bronchoprotection by tiotropium.

BACKGROUND AND PURPOSE: The bronchodilator tiotropium binds not only to its main binding site on the M3 muscarinic receptor but also to an allosteric site. Here, we have investigated the functional relevance of this allosteric binding and the potential contribution of this behaviour to interactions with long-acting ß-adrenoceptor agonists, as combination therapy with anticholinergic agents and ß-adrenoceptor agonists improves lung function in chronic obstructive pulmonary disease. EXPERIMENTAL APPROACH: ACh, tiotropium, and atropine binding to M3 receptors were modelled using molecular dynamics simulations. Contractions of bovine and human tracheal smooth muscle strips were studied. KEY RESULTS: Molecular dynamics simulation revealed extracellular vestibule binding of tiotropium, and not atropine, to M3 receptors as a secondary low affinity binding site, preventing ACh entry into the orthosteric binding pocket. This resulted in a low (allosteric binding) and high (orthosteric binding) functional affinity of tiotropium in protecting against methacholine-induced contractions of airway smooth muscle, which was not observed for atropine and glycopyrrolate. Moreover, antagonism by tiotropium was insurmountable in nature. This behaviour facilitated functional interactions of tiotropium with the ß-agonist olodaterol, which synergistically enhanced bronchoprotective effects of tiotropium. This was not seen for glycopyrrolate and olodaterol or indacaterol but was mimicked by the interaction of tiotropium and forskolin, indicating no direct ß-adrenoceptor-M3 receptor crosstalk in this effect. CONCLUSIONS AND IMPLICATIONS: We propose that tiotropium has two binding sites at the M3 receptor that prevent ACh action, which, together with slow dissociation kinetics, may contribute to insurmountable antagonism and enhanced functional interactions with ß-adrenoceptor agonists.

Cigarette smoke and non-neuronal cholinergic system in the airway epithelium of COPD patients.

Acetylcholine (ACh), synthesized by Choline Acetyl-Transferase (ChAT), exerts its physiological effects via mAChRM3 in epithelial cells. We hypothesized that cigarette smoke affects ChAT, ACh, and mAChRM3 expression in the airways from COPD patients promoting airway disease. ChAT, ACh, and mAChRM3 were assessed: "ex vivo" in the epithelium from central and distal airways of COPD patients, Healthy Smoker (S) and Healthy Subjects (C), and "in vitro" in bronchial epithelial cells stimulated with cigarette smoke extract (CSE). In central airways, mAChRM3, ChAT, and ACh immunoreactivity was significantly higher in the epithelium from S and COPD than in C subjects. mAChRM3, ChAT, and ACh score of immunoreactivity was high in the metaplastia area of COPD patients. mAChRM3/ChAT and ACh/ChAT co-localization of immunoreactivity was observed in the bronchial epithelium from COPD. In vitro, CSE stimulation significantly increased mAChRM3, ChAT, and ACh expression and mAChRM3/ChAT and ACh/ChAT co-localization in 16HBE and NHBE, and increased 16HBE proliferation. Cigarette smoke modifies the levels of mAChMR3, ChAT expression, and ACh production in bronchial epithelial cells from COPD patients. Non-neuronal components of cholinergic system may have a role in the mechanism of bronchial epithelial cell proliferation, promoting alteration of normal tissue, and of related pulmonary functions.

Effects of tiotropium bromide on airway hyperresponsiveness and inflammation in mice exposed to organic dust.

INTRODUCTION: Acute exposure to organic dust (OD) in pig barns induces intense airway inflammation with neutrophilia and hyperresponsiveness. This reaction is likely associated with increased cholinergic activity. Therefore, the involvement of cholinergic mechanisms in the reaction to acute exposure of OD was investigated in mice using the long-acting muscarinic antagonist tiotropium. METHODS: BALB/c mice received tiotropium (2-200 ng) intranasally on day 1 of the study. On days 2-4, mice received vehicle or OD (25 µg) intranasally. Airway hyperresponsiveness to methacholine was assessed 24 h following the last OD exposure. Bronchoalveolar lavage (BAL) fluid, lung tissue and blood were collected for analyses. RESULTS: Organic dust elevated airway responsiveness to methacholine compared with controls (PBS) assessed as Newtonian resistance (1.5 ±â€¯0.1 vs 0.9 ±â€¯0.1 cm H2O x s/mL), tissue damping (12.4 ±â€¯1.4 vs 8.9 ±â€¯0.9 cm H2O∙s/mL) and tissue elastance (41.1 ±â€¯5.3 vs 27.2 ±â€¯2.5 cm H2O∙s/mL). Tiotropium (200 ng) decreased the Newtonian resistance and tissue damping after exposure to PBS or OD. Organic dust exposure increased inflammatory cells in BAL fluid by almost 400%, mainly due to neutrophil influx, which was unaffected by tiotropium. Organic dust increased levels of mainly Th1 mediators. Tiotropium treatment attenuated OD-induced release of IL-2, IL-4 and IL-6. CONCLUSIONS: Tiotropium decreased the OD-induced increase of specific cytokines without influencing the OD-induced increase of airway responsiveness and neutrophil infiltration into the lungs. We conclude that the cholinergic pathway contributes to the pro-inflammatory effects caused by inhalation of OD from pig barns.

The effect of tiotropium in combination with olodaterol on house dust mite-induced allergic airway disease.

One of the major goals of asthma therapy is to maintain asthma control and prevent acute exacerbations. Long-acting bronchodilators are regularly used for the treatment of asthma patients and in clinical studies the anti-cholinergic tiotropium has recently been shown to reduce exacerbations in patients with asthma. So far it is unclear how tiotropium exerts this effect. For this purpose, we designed an allergen-driven rechallenge model of allergic airway inflammation in mice, to assess the effectiveness of tiotropium and the long-acting ß-2 adrenoceptor agonist olodaterol on allergen-induced exacerbations of airway disease. Female C57BL/6J mice were sensitized intranasally (i.n.) with 1 µg of house dust mite (HDM) extract followed by a challenge regime (5 consecutive days 10 µg HDM extract i.n.) after one week. Mice were exposed to a secondary challenge five weeks after sensitization and were treated i.n. with different concentrations of tiotropium or olodaterol (1, 10 and 100 µg/kg) or a combination thereof (10 µg/kg each) prior to and during the secondary challenge period. Three days after the last challenge, bronchoalveolar lavage (BAL) fluid and lung tissue were collected for flow cytometry and histologic analysis, respectively. Secondary challenge with HDM extract strongly induced allergic airway disease reflected by inflammatory cell infiltration and goblet cell metaplasia. Treatment with tiotropium, but not with olodaterol reduced tissue inflammation and goblet cell metaplasia in a dose-dependent manner. The combination of tiotropium and olodaterol was more effective in significantly reducing tissue inflammation compared to tiotropium treatment alone, and also led to a decrease in BAL cell counts. These data suggest that in a model of relapsing allergic airway disease tiotropium directly prevents exacerbations by reducing inflammation and mucus production in the airways. In addition, the combination of tiotropium and olodaterol exerts synergistic effects.

Effect of tiotropium on COPD exacerbations: A systematic review.

BACKGROUND: Exacerbation frequency is related to disease progression, quality of life, and prognosis in COPD. Earlier diagnosis, along with interventions aimed at preventing exacerbations and delaying progression, may help reduce the global burden of disease. Long-acting inhaled bronchodilators are effective at maintaining symptom relief and are recommended as first-choice therapy for more symptomatic patients and those at risk of exacerbation. METHODS: As prevention of exacerbations is a priority goal in COPD management and a number of different long-acting bronchodilators are available, we conducted a systematic review of exacerbation data from randomized controlled trials (published January 2000 to May 2014) comparing the effect of tiotropium versus placebo and/or other maintenance therapies. RESULTS: Exacerbations were a primary endpoint in 12 publications (five studies: four comparing tiotropium with placebo; one with active comparator) and a secondary endpoint in 17 publications (seven studies: six comparing tiotropium with placebo; one with active comparator). Overall, tiotropium was associated with a longer time to first exacerbation event and fewer exacerbations (including severe exacerbations/hospitalizations) compared with placebo and long-acting ß2-agonists. Tiotropium also showed similar efficacy to glycopyrronium and a fixed long-acting muscarinic antagonist/long-acting ß2-agonist combination (glycopyrronium/indacaterol), although not all studies were powered to demonstrate differences in exacerbation outcomes. Exacerbation outcomes were comparable with both formulations of tiotropium (HandiHaler(®) 18 µg/Respimat(®) 5 µg). CONCLUSIONS: The results of this comprehensive systematic review demonstrate tiotropium is beneficial in reducing exacerbation risk versus placebo or other maintenance treatments.

The pharmacological rationale for combining muscarinic receptor antagonists and ß-adrenoceptor agonists in the treatment of airway and bladder disease.

Muscarinic receptor antagonists and ß-adrenoceptor agonists are used in the treatment of obstructive airway disease and overactive bladder syndrome. Here we review the pharmacological rationale for their combination. Muscarinic receptors and ß-adrenoceptors are physiological antagonists for smooth muscle tone in airways and bladder. Muscarinic agonism may attenuate ß-adrenoceptor-mediated relaxation more than other contractile stimuli. Chronic treatment with one drug class may regulate expression of the target receptor but also that of the opposing receptor. Prejunctional ß2-adrenoceptors can enhance neuronal acetylcholine release. Moreover, at least in the airways, muscarinic receptors and ß-adrenoceptors are expressed in different locations, indicating that only a combined modulation of both systems may cause dilatation along the entire bronchial tree. While all of these factors contribute to a rationale for a combination of muscarinic receptor antagonists and ß-adrenoceptor agonists, the full value of such combination as compared to monotherapy can only be determined in clinical studies.

Tiotropium bromide inhibits relapsing allergic asthma in BALB/c mice.

Recurrent relapses of allergic lung inflammation in asthmatics may lead to airway remodeling and lung damage. We tested the efficacy of tiotropium bromide, a selective long-acting, muscarinic receptor antagonist as an adjunct therapy in relapses of allergic asthma in mice. We compared the effectiveness of local intranasal administration of tiotropium and dexamethasone in acute and relapsing allergic asthma in BALB/c mice. Although tiotropium at low doses is a potent bronchodilator, we tested higher doses to determine effectiveness on inflammation and mucus hypersecretion. A 5-day course of twice daily intranasal tiotropium or dexamethasone (1 mg/kg (b.w.)) suppressed airway eosinophils by over 87% during disease initiation and 88% at relapse compared to vehicle alone. Both drugs were comparable in their capacity to suppress airway and parenchymal inflammation and mucus hypersecretion, though tiotropium was better than dexamethasone at reducing mucus secretion during disease relapse. Despite treatment with either drug, serum antigen-specific IgE or IgG1 antibody titres remained unchanged. Our study indicates that tiotropium at higher doses than required for bronchodilation, effectively suppresses inflammation and mucus hypersecretion in the lungs and airways of mice during the initiation and relapse of asthma. Tiotropium is currently not approved for use in asthma. Clinical studies have to demonstrate the efficacy of tiotropium in this respiratory disease.

Molecular basis for the long duration of action and kinetic selectivity of tiotropium for the muscarinic M3 receptor.

Antagonizing the human M3 muscarinic receptor (hM3R) over a long time is a key feature of modern bronchodilating COPD drugs aiming at symptom relief. The long duration of action of the antimuscarinic drug tiotropium and its kinetic subtype selectivity over hM2R are investigated by kinetic mapping of the binding site and the exit channel of hM3R. Hence, dissociation experiments have been performed with a set of molecular matched pairs of tiotropium on a large variety of mutated variants of hM3R. The exceedingly long half-life of tiotropium (of more than 24 h) is attributed to interactions in the binding site; particularly a highly directed interaction of the ligands' hydroxy group with an asparagine (N508(6.52)) prevents rapid dissociation via a snap-lock mechanism. The kinetic selectivity over hM2R, however, is caused by differences in the electrostatics and in the flexibility of the extracellular vestibule. Extensive molecular dynamics simulations (several microseconds) support experimental results.

In vitro validation of a Respimat® adapter for delivery of inhaled bronchodilators during mechanical ventilation.

BACKGROUND: Inhaled bronchodilators are frequently used in patients with chronic obstructive pulmonary disease (COPD). However, there has been no efficient way to administer the long-acting anticholinergic tiotropium to mechanically ventilated patients. The aim of this in vitro study was to compare the fine particle dose (FPD) output of a specifically designed adapter with other accessory devices for the delivery of bronchodilators using the Respimat® (RMT) inhaler by simulating the specific inhalation flow profiles of patients with COPD. METHODS: Using characteristic flow profiles from COPD patients being weaned off mechanical ventilation, an in vitro study was performed analyzing the FPD achieved with different accessory devices (connectors, spacers, AeroTrachPlus valved holding chamber), which can be used to deliver drugs from pressurized metered dose inhalers (pMDI) and RMT inhalers to artificial airways. Fenoterol pMDI, tiotropium RMT, and a fixed-dose combination of salbutamol and ipratropium delivered by pMDI or RMT, were used as bronchodilators. Aerosols were collected by a next-generation impactor. RESULTS: The RMT inhaler, combined with a new in-line adapter, was superior to other inhaler device connector or spacer combinations in FPD delivery during simulated mechanical ventilation (p<0.01). The outcome with the RMT inhaler/RMT adapter combination during simulation of mechanical ventilation was comparable to the measurements with the RMT/AeroTrachPlus valved holding chamber during simulation of spontaneous breathing. The delivery rates of the RMT adapter were not significantly affected by the administered bronchodilators or by the type of artificial airway (endotracheal or tracheostomy tube) employed. CONCLUSIONS: The RMT inhaler combined with the prototype in-line adapter was better than the other accessory device combinations in fine particle deposition of inhaled bronchodilators during mechanical ventilation. Further research is required to determine the clinical relevance of these in vitro findings.

Acetylcholine mediates the release of IL-8 in human bronchial epithelial cells by a NFkB/ERK-dependent mechanism.

Acetylcholine may play a role in cell activation and airway inflammation. We evaluated the levels of both mRNA and protein of muscarinic M(1), M(2), M(3) receptors in human bronchial epithelial cell line (16HBE). 16HBE cells were also stimulated with acetylcholine and extracellular signal-regulated kinase1/2 (ERK1/2) and NFkB pathway activation as well as the IL-8 release was assessed in the presence or absence of the inhibitor of Protein-kinase (PKC) (GF109203X), of the inhibitor of mitogenic activated protein-kinase kinase (MAPKK) (PDO9805), of the inhibitor of kinaseB-alpha phosphorilation (pIkBalpha) (BAY11-7082), and of muscarinic receptor antagonists tiotropium bromide, 4-Diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), telenzepine, gallamine. Additionally, we tested the IL-8-mediated neutrophil chemotactic activity of 16HBE supernatants stimulated with acetylcholine in the presence or absence of tiotropium. 16HBE cells expressed both protein and mRNA for muscarinic M(3), M(2) and M(1) receptors with levels of muscarinic M(3) receptor>muscarinic M(1) receptor>muscarinic M(2) receptor. Acetylcholine (10 microM) significantly stimulated ERK1/2 and NFkB activation as well as IL-8 release in 16HBE cells when compared to basal values. Furthermore, while the use of tiotropium, 4-DAMP, GF109203X, PDO98059, BAY11-7082 completely abolished these events, the use of telenzepine and gallamine were only partially able to downregulate these effects. Additionally, acetylcholine-mediated IL-8 release from 16HBE cells significantly increased chemotaxis toward neutrophils and this effect was blocked by tiotropium. In conclusion, acetylcholine activates the release of IL-8 from 16HBE involving PKC, ERK1/2 and NFkB pathways via muscarinic receptors, suggesting that it is likely to contribute to IL-8 related neutrophilic inflammatory disorders in the airway. Thus, muscarinic antagonists may contribute to control inflammatory processes in airway diseases.