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.