Introduction
Diabetic kidney disease (DKD) is a prevalent metabolic disease and is the most common cause of end stage renal disease (ESRD) (Fu et al., 2019). Despite existing therapies that target hypertension and hyperglycemia, these treatments only slow the progression to ESRD. Thus, there is a need for the identification and development of new pharmacological therapeutics to treat DKD.
In type 2 diabetes, early hyperglycemia and glomerular hyperfiltration affect renal glomerular and proximal tubular function. Hyperglycemia exposes the proximal tubule to increased amounts of filtered glucose, which in turn leads to increased glucose reabsorption (Vallon, 2011). The increased tubular glucose load results in a number of pathophysiological changes: proximal tubule growth, upregulation of sodium-glucose cotransporter 2 (SGLT2), inflammation, mitochondrial dysfunction and eventually leading to tubulointerstitial fibrosis (Abbate & Remuzzi, 1999; Bohle et al., 1991; Cleveland et al., 2020; Forbes & Thorburn, 2018; Huang & Preisig, 2000; Vallon & Verma, 2021). Although there are clear and distinct changes in proximal tubular function in DKD, the mechanisms underlying these changes are understudied.
The kidney is a highly metabolic organ that relies heavily on mitochondrial oxygen consumption to account for the energy requirements of tubular reabsorption (Bhargava & Schnellmann, 2017; Lynch et al., 2018). Mitochondrial dysfunction has been identified as a key event in the early stages of hyperglycemia leading to disease progression. Renal mitochondrial dysfunction encompasses multiple functional changes. Studies have shown that expression and activity of the master regulator of mitochondrial biogenesis (MB) peroxisome proliferator-activated receptor gamma co-activator 1 alpha (PGC1α) is downregulated in proximal tubules of diabetic animals, leading to disease progression (Lee et al., 2017).
In addition, studies report that mitochondrial dynamics is altered in diabetic db/db mice and in glomerular podocytes exposed to high glucose (Ayanga et al., 2016; Galvan et al., 2019). These changes in mitochondrial dynamics were mediated by the mitochondrial fission protein dynamin-like protein 1 (Drp1). When Drp1 was knocked down or pharmacologically inhibited, mitochondrial function was restored. Importantly, restoration of mitochondrial dynamics and function further led to significant improvement of hallmark features of DKD including glomerular scarring, albuminuria and mesangial matrix expansion. It has also been reported that Mfn1 expression decreases in response to high glucose, although the extent of knowledge regarding Mfn1 in DKD is limited (Audzeyenka et al., 2021). These studies provide evidence that mitochondrial function is altered in response to glucose and improving aspects of mitochondrial dysfunction such as mitochondrial dynamics can be an effective therapeutic strategy in DKD.
We previously demonstrated that in RPTC exposed to high glucose, phosphorylation of the mitochondrial fission protein Drp1 was increased and expression the mitochondrial fusion protein Mfn1 was decreased, indicating that there is an imbalance in RPTC mitochondrial dynamics in the presence of glucose (Cleveland et al., 2020). Interestingly, the same effects were observed in renal cortical tissue of early diabetic db/db mice. Despite these clear and distinct alterations in mitochondrial dynamics proteins, the signaling mechanisms underlying these effects remain unknown. Studies have identified that Drp1 phosphorylation in hyperglycemia is mediated by Rho-associated protein kinase 1 (ROCK1) (W. Wang et al., 2012). Furthermore, separate studies show that Ras homolog family member A (RhoA) is responsible for ROCK1 activation and subsequent Drp1 phosphorylation (Brand et al., 2018). Little is known about the role of Mfn1 and its associated signaling pathways in high glucose. However, it has been shown that MEK1/2/ERK1/2 regulates Mfn1 function (Pyakurel et al., 2015). In addition, it has been demonstrated that Raf, the upstream activator of MEK1/2 is also upregulated in the presence of glucose (Trumper et al., 2005).
Our previous study showed that altered Drp1 phosphorylation and Mfn1 expression in diabetic db/db mice as well as in RPTC exposed to high glucose was restored by treatment with the β2-adrenoceptor agonist formoterol. However, the mechanisms as to how formoterol activation of the β2-adrenoceptor regulates mitochondrial dynamics have yet to be determined. In separate studies, we also demonstrated that formoterol promotes recovery from acute kidney injury (AKI) by stimulating MB (Jesinkey et al., 2014; Wills et al., 2012). Despite the well-known classical pathway of β2-adrenoceptor activation, defined by Gαs stimulation of adenylyl cyclase (AC) and cAMP production, we discovered that formoterol signals through Gβγ to activate Akt/eNOS/sGC/PGC1α to induce MB (Cameron et al., 2017). Based on these findings, our hypothesis was formoterol signals through the Gβγ subunit of the β2-adrenoceptor to regulate RhoA/ROCK1/Drp1 and Raf/MEK1/2/ERK1/2/Mfn1 to restore the imbalance between mitochondrial fission and fusion in DKD.