Cleveland Kristan H, Schnellmann Rick G
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, AZ, United States.
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, AZ, United States; Southern Arizona VA Health Care System, Tucson, AZ, United States; Southwest Environmental Health Science Center, University of Arizona, Tucson, AZ, United States.
Biochem Pharmacol. 2023 Mar;209:115436. doi: 10.1016/j.bcp.2023.115436. Epub 2023 Jan 30.
Mitochondrial dysfunction drives the development and progression of diabetic kidney disease (DKD). Previously, we discovered that the β-adrenergic receptor (AR) agonist formoterol regulates mitochondrial dynamics in the hyperglycemic renal proximal tubule. The goal of this study was to identify signaling mechanisms through which formoterol restores the mitochondrial fission/fusion proteins Drp1 and Mfn1. Using primary renal proximal tubule cells (RPTC), the effect of chronic high glucose on RhoA/ROCK1/Drp1 and Raf/MEK1/2/ERK1/2/Mfn1 signaling was determined. In glucose-treated RPTC, RhoA became hyperactive, leading to ROCK1-induced activation of Drp1. Treatment with formoterol and/or pharmacological inhibitors targeting RhoA, ROCK1 and Drp1 blocked RhoA and Drp1 hyperactivity. Inhibiting this pathway also restored maximal mitochondrial respiration. By preventing Gβγ signaling with gallein, we determined that formoterol signals through the Gβγ subunit of the β-AR to restore RhoA and Drp1. Furthermore, formoterol restored this pathway by blocking binding of RhoA with the guanine nucleotide exchange factor p114RhoGEF. Formoterol also restored the mitochondrial fusion protein Mfn1 through a second Gβγ-dependent mechanism composed of Raf/MEK1/2/ERK1/2/Mfn1. Glucose-treated RPTC exhibited decreased Mfn1 activity, which was restored with formoterol. Pharmacological inhibition of Gβγ, Raf and MEK1/2 also restored Mfn1 activity. We demonstrate that glucose promotes the interaction between RhoA and p114RhoGEF, leading to increased RhoA and ROCK1-mediated activation of Drp1, and decreases Mfn1 activity through Raf/MEK1/2/ERK1/2. Formoterol restores these pathways and mitochondrial function in response to elevated glucose by activating separate yet integrative pathways that promote mitochondrial biogenesis, decreased fission and increased fusion in RPTC, further supporting its potential as a therapeutic for DKD.
线粒体功能障碍驱动糖尿病肾病（DKD）的发生和发展。此前，我们发现β-肾上腺素能受体（AR）激动剂福莫特罗可调节高血糖肾近端小管中的线粒体动力学。本研究的目的是确定福莫特罗恢复线粒体裂变/融合蛋白Drp1和Mfn1的信号传导机制。使用原代肾近端小管细胞（RPTC），确定了慢性高糖对RhoA/ROCK1/Drp1和Raf/MEK1/2/ERK1/2/Mfn1信号传导的影响。在葡萄糖处理的RPTC中，RhoA变得过度活跃，导致ROCK1诱导的Drp1激活。用福莫特罗和/或靶向RhoA、ROCK1和Drp1的药理抑制剂处理可阻断RhoA和Drp1的过度活跃。抑制该途径还可恢复最大线粒体呼吸。通过用加兰他敏阻断Gβγ信号传导，我们确定福莫特罗通过β-AR的Gβγ亚基发出信号以恢复RhoA和Drp1。此外，福莫特罗通过阻断RhoA与鸟嘌呤核苷酸交换因子p114RhoGEF的结合来恢复该途径。福莫特罗还通过由Raf/MEK1/2/ERK1/2/Mfn1组成的第二种Gβγ依赖性机制恢复线粒体融合蛋白Mfn1。葡萄糖处理的RPTC表现出Mfnl活性降低，而福莫特罗可使其恢复。对Gβγ、Raf和MEK1/2的药理抑制也恢复了Mfn1活性。我们证明葡萄糖促进RhoA与p114RhoGEF之间的相互作用，导致RhoA和ROCK1介导的Drp1激活增加，并通过Raf/MEK1/2/ERK1/2降低Mfn1活性。福莫特罗通过激活促进RPTC中线粒体生物发生、减少裂变和增加融合的独立但相互整合的途径，来响应葡萄糖升高而恢复这些途径和线粒体功能，进一步支持了其作为DKD治疗药物的潜力。
