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Protein kinase D2 regulates epithelial sodium channel activity and aldosterone non-genomic responses in renal cortical collecting duct cells

Thomas W, Dooley R, Quinn S, Robles MY, Harvey BJ

Steroids, 2020 03;155:108553.
PMID: 31836481 DOI: 10.1016/j.steroids.2019.108553

Protein kinase D2 (PKD2) is a serine/threonine protein kinase which plays an important role in vesicle fission at the trans-Golgi network (TGN) to coordinate subcellular trafficking with gene expression. We found that in the rat kidney, PKD2 is specifically expressed in collecting duct principal cells predominantly at the apical membrane and with lower basal expression in cytosolic compartments. When rats were maintained on a Na+ depleted diet (<0.87 mmol Na+/kg) to increase plasma aldosterone levels, PKD2 became internalized to a cytoplasmic compartment. Treatment of murine M1 cortical collecting duct (M1-CCD) cells with aldosterone (10 nM) promoted PKD2 co-localization with the trans-Golgi network within 30 min. PKD2 underwent autophosphorylation at Ser876 within 10 min of aldosterone treatment and remained phosphorylated (active) for at least 24 h. A stable PKD2 shRNA knock-down (PKD2 KD) M1-CCD cell line was developed to study the role of PKD2 in epithelial Na+ channel (ENaC) trafficking and transepithelial Na+ transport (SCC) in epithelial monolayers grown in Ussing chambers. The PKD2 KD cells developed transepithelial resistance with kinetics equivalent to wild-type cells, however the transepithelial voltage and Na+ current were significantly elevated in PKD2 knock-down CCD epithelia. The higher basal SCC was due to increased ENaC activity. Aldosterone treatment for 24 h resulted in a decline in ENaC activity in the PKD2 KD cells as opposed to the increase observed in the wild-type cells. The paradoxical inhibition of SCC by aldosterone in PKD2 KD epithelium was attributed to a reduction in ENaC current and lower membrane abundance of ENaC, demonstrating that PKD2 plays a critical tonic role in ENaC trafficking and channel subunit stability. The rapid activation of PKD2 by aldosterone is synergistic with the transcriptional activity of MR and contributes to increased ENaC activity.
Fulltext Tolerability, safety and intermediary pharmacological effects of cilostazol and isosorbide mononitrate, alone and combined, in patients with lacunar ischaemic stroke: The LACunar Intervention-1 (LACI-1) trial, a randomised clinical trial

Blair GW, Appleton JP, Flaherty K, Doubal F, Sprigg N, Dooley R, et al.

EClinicalMedicine, 2019 04 24;11:34-43.
PMID: 31317131 DOI: 10.1016/j.eclinm.2019.04.001

Background: Lacunar stroke, a frequent clinical manifestation of small vessel disease (SVD), differs pathologically from other ischaemic stroke subtypes and has no specific long-term secondary prevention. Licenced drugs, isosorbide mononitrate (ISMN) and cilostazol, have relevant actions to prevent SVD progression.
Methods: We recruited independent patients with clinically confirmed lacunar ischaemic stroke without cognitive impairment to a prospective randomised clinical trial, LACunar Intervention-1 (LACI-1). We randomised patients using a central web-based system, 1:1:1:1 with minimisation, to masked ISMN 25 mg bd, cilostazol 100 mg bd, both ISMN and cilostazol started immediately, or both with start delayed. We escalated doses to target over two weeks, sustained for eight weeks. Primary outcome was the proportion achieving target dose. Secondary outcomes included symptoms, safety (haemorrhage, recurrent vascular events), cognition, haematology, vascular function, and neuroimaging. LACI-1 was powered (80%, alpha 0.05) to detect 35% (90% versus 55%) difference between the proportion reaching target dose on one versus both drugs at 55 patients. Registration ISRCTN12580546.
Findings: LACI-1 enrolled 57 participants between March 2016 and August 2017: 18 (32%) females, mean age 66 (SD 11, range 40-85) years, onset-randomisation 203 (range 6-920) days. Most achieved full (64%) or over half (87%) dose, with no difference between cilostazol vs ISMN, single vs dual drugs. Headache and palpitations increased initially then declined similarly with dual versus single drugs. There was no between-group difference in BP, pulse-wave velocity, haemoglobin or platelet function, but pulse rate was higher (mean difference, MD, 6.4, 95%CI 1.2-11.7, p = 0.02), platelet count higher (MD 35.7, 95%CI 2.8, 68.7, p = 0.03) and white matter hyperintensities reduced more (Chi-square p = 0.007) with cilostazol versus no cilostazol.
Interpretation: Cilostazol and ISMN are well tolerated when the dose is escalated, without safety concerns, in patients with lacunar stroke. Larger trials with longer term follow-up are justified.
Funding: Alzheimer's Society (AS-PG-14-033).
Preventing cognitive decline and dementia from cerebral small vessel disease: The LACI-1 Trial. Protocol and statistical analysis plan of a phase IIa dose escalation trial testing tolerability, safety and effect on intermediary endpoints of isosorbide mononitrate and cilostazol, separately and in combination

Blair GW, Appleton JP, Law ZK, Doubal F, Flaherty K, Dooley R, et al.

Int J Stroke, 2018 07;13(5):530-538.
PMID: 28906205 DOI: 10.1177/1747493017731947

Rationale The pathophysiology of most lacunar stroke, a form of small vessel disease, is thought to differ from large artery atherothrombo- or cardio-embolic stroke. Licensed drugs, isosorbide mononitrate and cilostazol, have promising mechanisms of action to support their testing to prevent stroke recurrence, cognitive impairment, or radiological progression after lacunar stroke. Aim LACI-1 will assess the tolerability, safety, and efficacy, by dose, of isosorbide mononitrate and cilostazol, alone and in combination, in patients with ischemic lacunar stroke. Sample size A sample of 60 provides 80+% power (significance 0.05) to detect a difference of 35% (90% versus 55%) between those reaching target dose on one versus both drugs. Methods and design LACI-1 is a phase IIa partial factorial, dose-escalation, prospective, randomized, open label, blinded endpoint trial. Participants are randomized to isosorbide mononitrate and/or cilostazol for 11 weeks with dose escalation to target as tolerated in two centers (Edinburgh, Nottingham). At three visits, tolerability, safety, blood pressure, pulse wave velocity, and platelet function are assessed, plus magnetic resonance imaging to assess cerebrovascular reactivity in a subgroup. Study outcomes Primary: proportion of patients completing study achieving target maximum dose. Secondary symptoms whilst taking medications; safety (hemorrhage, recurrent vascular events, falls); blood pressure, platelet function, arterial stiffness, and cerebrovascular reactivity. Discussion This study will inform the design of a larger phase III trial of isosorbide mononitrate and cilostazol in lacunar stroke, whilst providing data on the drugs' effects on vascular and platelet function. Trial registration ISRCTN (ISRCTN12580546) and EudraCT (2015-001953-33).

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