Discussion
This study investigated the signaling pathways associated with altered mitochondrial dynamics in RPTC exposed to high glucose. Increased expression and activation of Drp1 has been implicated in multiple cell types along the nephron and contributes to mitochondrial dysfunction in models of DKD and RPTC (Ayanga et al., 2016; Cleveland et al., 2020; Galvan et al., 2019; S. Wang et al., 2012). Prior studies have identified that ROCK1 is an activator of Drp1 (W. Wang et al., 2012). Separate studies have identified that RhoA also leads to increased Drp1 phosphorylation and translocation to the mitochondria in a ROCK1-dependent manner (Brand et al., 2018). Aside from RhoA/ROCK1, Drp1 is also phosphorylated by protein kinase D (PKD) at Ser637 to initiate mitochondrial fission (Jhun et al., 2018). In addition, Drp1 is also activated by protein kinase A (PKA)-induced phosphorylation of Ser637, although phosphorylation by PKA has been demonstrated to have an inhibitory effect on Drp1 (Chang & Blackstone, 2007).
The RhoA/ROCK1/Drp1 pathway is hyperactivated in RPTC treated with high glucose. Using inhibitors of these proteins, we confirmed this signaling pathway and determined that formoterol acts through the Gβγ subunit of the β2-adrenoceptor to inhibit p114RhoGEF interaction with RhoA. By preventing this interaction, there was a restoration of both RhoA and Drp1 activity. Ultimately, formoterol blocks glucose-induced DRP 1 activity Despite this finding, it is still unclear exactly how Gβγ blocks the interaction between p114RhoGEF and RhoA.
As previously reported, high glucose decreases maximal mitochondrial respiration (Cleveland et al., 2020). Using pharmacological inhibitors targeting RhoA, ROCK1 and Drp1, maximal mitochondrial respiration was restored, indicating that in addition to its role in mitochondrial fission, Drp1 may also play a role in regulating mitochondrial respiration. A study evaluating the effects of Drp1 inhibition on mitochondrial function found that inhibition of endogenous Drp1 suppressed maximal respiration. Furthermore, the authors showed that Drp1 is responsible for maintaining mitochondrial respiration and bioenergetics independently of its role in mitochondrial fission (Zhang et al., 2017), indicating that Drp1 regulates mitochondrial function in addition to its role in maintaining mitochondrial morphology.
Prior work has noted that components of the proposed Mfn1 pathway, such as Raf, MEK1/2 and ERK1/2 are upregulated in response to elevated glucose (Duan & Cobb, 2010; Khoo & Cobb, 1997; Trumper et al., 2005). Pyakurel et al. demonstrated that MEK1/2/ERK1/2 phosphorylates Mfn1 to modulate its activity as a mechanism of apoptosis regulation (Pyakurel et al., 2015). Based on these studies, there is evidence linking MEK1/2/ERK1/2 to Mfn1 activation. Despite this knowledge, the signaling pathway has not yet been elucidated in renal cell types exposed to hyperglycemic conditions. High glucose decreased Mfn1 and pharmacological inhibitors of Raf and MEK1/2 restored Mfn1 activity. Importantly, when Gβγ was blocked with gallein, formoterol lost its effect on Mfn1, indicating that this mitochondrial fusion pathway is restored by activating Gβγ. Our previous study showed that formoterol induces MB through Gβγ-induced phosphorylation of Akt. However, this study demonstrates that in RPTC, neither glucose nor formoterol effects the phosphorylation status of Akt, indicating that the pathways through which formoterol restores mitochondrial fission/fusion are separate from that which regulates MB. It is important to note that the present study does not determine exactly how formoterol activation of Gβγ directly leads to decreased Raf activation to ultimately restore Mfn1. However, it has been previously reported that both Gα and Gβγ can regulate MAPK signaling pathways (Goldsmith & Dhanasekaran, 2007; Ito et al., 1995), further indicating that the resulting outcome of G-protein subunit activation likely relies on context and stimulus specificity.
MB plays an important role maintaining mitochondrial homeostasis and in regulating cellular metabolism. Pharmacologically activating MB has shown potential as a therapeutic strategy (Whitaker et al., 2016). It has previously been determined that formoterol activation of Gβγ leads to PI3K-dependent activation of Akt/eNOS/sGC in RPTC (Cameron et al., 2017), and activation of this pathway leads to increased PGC1α and MB. In this study, we show that that in addition to its role in activating Akt/eNOS/sG to induce MB, formoterol also restores mitochondrial fission through p114/RhoA/ROCK1/Drp1 and mitochondrial fusion through Raf/MEK1/2/ERK1/2/Mfn1 through Gβγ-dependent mechanisms. Although formoterol induces three separate and distinct pathways, they are integrated and work simultaneously to restore MB and mitochondrial homeostasis in RPTC in response to high glucose injury (graphical abstract). It is widely known that β-arrestins (β-arr1) 1 and 2 are required for ERK1/2 activation via scaffolding of Raf/MEK1/2/ERK1/2. However, a study by O’Hayre et al. demonstrated that β-arr1 and β-arr2 are dispensable for β2-AR-dependent ERK activation (O’Hayre et al., 2017). Rather, the authors demonstrated that β2-ARs signal through Gαs and Gβγ to activate the tyrosine kinase Src and the guanine nucleotide exchange factor SOS to activate Raf, MEK and ERK. These findings support our results which demonstrate that formoterol signals through Gβγ to restore mitochondrial homeostasis.
While current therapies for DKD are effective at targeting hypertension and hyperglycemia, there are limited and indirect drugs that decrease disease progression. A common property of these therapeutics lies in their ability to modulate mitochondrial function. Studies evaluating the effects of angiotensin receptor type 1 and type 2 receptor (AT1R and AT2R) blockers showed that olmesartan restored altered expression of TCA cycle enzymes and the superoxide generating enzyme Nox2 (Vazquez-Medina et al., 2013). Another antagonist, losartan, provided renal mitochondrial protection from oxidative injury (Katyare & Satav, 2005). Interestingly, studies evaluating the effects of anti-hyperglycemic agents on mitochondrial effects showed that the SGLT2 inhibitor dapagliflozin also demonstrated a protective effect on mitochondrial function and stimulated PGC1α (He et al., 2022). These studies provide evidence that mitochondrial dysfunction is a driving factor in the development and progression of DKD and support the hypothesis that mitochondrial therapies can improve both renal function and hallmark features of DKD.
Formoterol has shown to be a promising therapeutic for the treatment of several kidney diseases. In addition to its beneficial effects on recovery from AKI, it has also been demonstrated that formoterol accelerates podocyte recovery from glomerular injury by inducing MB (Arif et al., 2019), providing evidence that improving mitochondrial function has a beneficial therapeutic effect on multiple renal diseases. Since formoterol is already an FDA-approved drug for the treatment of asthma, its use as a repurposed therapeutic for DKD would be both a time and cost-effective strategy. Although it has been demonstrated that formoterol can restore mitochondrial function leading to improved kidney function, all the mechanisms through which formoterol exerts these effects are still being uncovered. In this study, we show that formoterol signals through novel, distinct and separate yet integrated mechanistic pathways to restore mitochondrial homeostasis in RPTC.