Mitochondrial energetics in the kidney

The kidney requires a large number of mitochondria to remove waste from the blood and regulate fluid and electrolyte balance. Mitochondria provide the energy to drive these important functions and can adapt to different metabolic conditions through a number of signalling pathways (for example, mechanistic target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) pathways) that activate the transcriptional co-activator peroxisome proliferator-activated receptor-γ co-activator 1α (PGC1α), and by balancing mitochondrial dynamics and energetics to maintain mitochondrial homeostasis. Mitochondrial dysfunction leads to a decrease in ATP production, alterations in cellular functions and structure, and the loss of renal function. Persistent mitochondrial dysfunction has a role in the early stages and progression of renal diseases, such as acute kidney injury (AKI) and diabetic nephropathy, as it disrupts mitochondrial homeostasis and thus normal kidney function. Improving mitochondrial homeostasis and function has the potential to restore renal function, and administering compounds that stimulate mitochondrial biogenesis can restore mitochondrial and renal function in mouse models of AKI and diabetes mellitus. Furthermore, inhibiting the fission protein dynamin 1-like protein (DRP1) might ameliorate ischaemic renal injury by blocking mitochondrial fission.

As discussed, mitochondria produce ATP via the ETC. At steady state, when electrons are passed through the ETC to molecular oxygen, a low concentration of superoxide anions is generated from complex I and complex III. Although a low level of ROS, such as superoxide anions, is important for cell function, high concentrations are toxic to mitochondria and the cell 26 – 28 ( FIG. 2 ). For example, under oxidative stress, increased levels of ROS can cause breaks in mitochondrial DNA (mtDNA) that cause mutations in the next generation of mitochondria; breaks in mtDNA also negatively affect the efficiency of the ETC, causing a decrease in ATP production and damaging proteins and lipids 29 . ROS can also trigger apoptosis in the cell by causing the release of cytochrome c , leading to mitochondrial dysfunction 29 . Therefore, mitochondria have antioxidant defence systems to counteract the excessive formation of additional ROS. Superoxide dismutase 2 (SOD2), which converts superoxide anions to hydrogen peroxide and oxygen, is specific for mitochondria 30 . Moreover, the transcription of genes encoding antioxidant enzymes, such as SOD2, catalase and glutathione peroxidase, is activated by nuclear factor erythroid 2-related factor 2 (NRF2) in response to oxidative stress, providing a mechanism to prevent excessive ROS production 31 . The importance of these antioxidant systems is to maintain optimal ATP production and sustain mitochondrial function. Another important antioxidant defence mechanism involves glutathione. Glutathione is a tripeptide (γ-glutamyl-cysteinal-glycine) nucleophile that can exist in a reduced form (GSH), or in an oxidized form as glutathione disulfide (GSSG). Mitochondria contain their own pool of glutathione, mitochondrial glutathione (mGSH), which not only helps to decrease excessive ROS levels but also prevents the release of cytochrome c from the inner membrane 32 . mGSH directly interacts with superoxide anions and becomes oxidized to GSSG 33 . Glutathione peroxidase (GPX) is located in both the cytoplasm and the mitochondria and uses GSH to reduce hydrogen peroxide to water, resulting in GSSG as a by-product 34 . GSSG cannot exit the mitochondria and is converted back to mGSH by glutathione reductase, for reuse or for elimination from the mitochondria 33 . The conversion of GSSG to mGSH requires NADPH, allowing crosstalk between the mechanism that maintains mGSH levels and the pentose phosphate pathway that produces NADPH. Together, these mechanisms have a major role in preventing excessive levels of ROS, and sustaining mitochondrial function. Uncoupling proteins are a family of mitochondrial transport proteins that are located in the mitochondrial inner membrane 35 , 36 . They transport protons across the inner membrane to the mitochondrial matrix. Mitochondrial uncoupling protein 2 (UCP2) is expressed in the kidney and is activated by mitochondrial ROS and other stimuli. An increase in ROS formation in the mitochondria activates UCP2, dissipating the proton motive force as heat and, as a result, reducing ROS production 36 , 37 . As ROS production contributes to mitochondrial dysfunction in AKI and diabetic nephropathy, UCP2 has been explored in the kidney and in these disease states 38 . Studies investigating the role of UCP2 polymorphisms in the kidney that exacerbate disease in patients with diabetic nephro pathy reveal that UCP2 is a potential target for treatment 39 . Lack of UCP2 has also been shown to worsen tubular injury after induction of experimental AKI in mice 38 . These studies show the importance of UCP2 in the kidney as well as its role in attenuating excessive ROS production. Mechanisms also exist to sustain mitochondrial function under hypoxic conditions. The lack of oxygen under hypoxic conditions decreases ATP production and causes cell death. In normoxic conditions, hypoxia-inducible factor 1α (HIF1α) is degraded in the presence of oxygen and α-ketoglutarate, an intermediate of the TCA cycle 40 . However, under hypoxic conditions, HIF1α heterodimerizes with HIF1β to form a transcription factor that binds to a hypoxia response element (HRE) present in genes that encode glycolytic enzymes and glucose transporters in the kidney 41 . Hypoxic conditions also alter the composition of complex IV of the ETC in which, at physiological conditions, the regulatory subunit 1 predominates in the ETC; during hypoxia, regulatory subunit 2 predominates in complex IV, which increases the efficiency of the ETC 42 . Several studies have shown that increasing the efficiency of the ETC increases the production of mitochondrial ROS under hypoxic conditions, although the mechanism by which this occurs is still unclear 43 – 45 . The effects of oxidative stress and hypoxia on mitochondrial morphology and energetics are discussed below.

Mitochondrial homeostasis requires a balance between mitochondrial biogenesis, fission and fusion, and mitophagy — the selective removal of non-functional and damaged mitochondria from cells by autophagy. All of these processes work together to maintain mitochondrial energetics, that is, the optimal production of ATP in normoxic conditions and in altered metabolic conditions. Mitochondrial biogenesis Mitochondrial biogenesis, which produces new and functional mitochondria, increases ATP production in response to increasing energy demands. Mitochondrial biogenesis is regulated by a range of transcriptional co-activators and co-repressors 56 , 57 . One study has shown that PGC1α is a prominent regulator, at the transcriptional level, of oxidative phosphorylation, the TCA cycle and fatty acid metabolism in the kidney 58 . In that study, the investigators performed gene expression profiling of kidneys from control mice and nephron-specific inducible PPARGC1A -knockout (NiPKO) mice that had been fed a chow diet or high-fat diet (HFD). Using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database , they analysed transcripts from all four groups of mice, and found a decrease in transcripts related to oxidative phosphorylation, TCA cycle and glycolysis in chow-fed NiPKO mice and in HFD-fed NiPKO mice. This finding supports the idea that inactivation of PGC1α in the kidney reduces mitochondrial function and metabolism and subsequently decreases mitochondrial biogenesis. Overexpression of PGC1α can also mitigate mitochondrial dysfunction in vitro after oxidant exposure, further supporting a role for mitochondrial biogenesis in mitochondrial homeostasis 59 . The activation of peroxisome proliferator-activated receptors (PPARs) and oestrogen-related receptors (ERRs) also contributes to the regulation of mitochondrial biogenesis, sometimes by these receptors directly interacting with PGC1α 60 ( FIG. 4 ). PPARs and ERRs are nuclear receptors that can be activated by fatty acids and steroid hormones such as oestrogen, and they elicit a response by binding to specific DNA response elements through their DNA-binding domains 61 . PGC1α can directly bind to these nuclear receptors and co-activate the transcription of genes, the protein products of which are involved in oxidative phosphorylation and fatty acid oxidation 62 , 63 . PGC1α activation results in its translocation from the cytoplasm to the nucleus, allowing it to upregulate the transcription of genes that are important for mitochondrial homeostasis and ATP production 64 . Transcription programmes downstream of PGC1α include nuclear and mitochondrial genes, as well as those involved in signalling pathways that regulate mitochondrial biogenesis (reviewed elsewhere 65 – 67 ). As the activation or suppression of PGC1α is regulated by external stimuli and post-translational modifications, it can be considered to be a nutrient sensor in the kidney. The expression and regulation of PGC1α in the kidney is still being explored. However, much of what is known about the regulation of PGC1α was discovered in the injured kidney as a result of disease states, such as diabetic nephropathy, ischaemia–reperfusion injury (IRI), sepsis, and cisplatin-induced AKI. Findings in these disease states support a role for PGC1α in the recovery phase from these diseases and in restoring mitochondrial function, highlighting PGC1α as a therapeutic target. Exercise and insulin stimulate an increase in PPARGC1A expression in skeletal muscle and in the heart, whereas fasting increases PPARGC1A expression in the liver 65 , 68 . In brown fat and muscle cells, cold exposure activates PGC1α 65 . In cases of oxidative stress or nutrient depletion, the activation of mitochondrial biogenesis helps rescue mitochondria from apoptosis 69 , 70 . In general, if the cell is in need of more energy, PGC1α is activated by deacetylation, whereas PGC1α is inactivated by acetylation when energy levels are high 65 . In addition to AMPK and mTOR, other energy sensing pathways that stimulate mitochondrial bio genesis include those involving sirtuins, cAMP and cyclic guanosine monophosphate (cGMP) ( FIG. 4 ). Sirtuin 1 (SIRT1) and SIRT3 are protein deacetylases that have a role in a variety of mitochondrial processes, including the ETC, TCA cycle, fatty acid oxidation, redox homeostasis and mitochondrial biogenesis 71 . SIRT1 activity is activated by NAD + , after which it activates downstream targets such as PGC1α 64 . SIRT3 is mitochondria- specific and can be activated to stimulate mitochondrial biogenesis 72 . The stimulation of adenylyl cyclase results in an increase in cAMP, which activates protein kinase A (PKA) that in turn phosphorylates cyclic AMP-responsive element-binding protein (CREB) 65 , 73 . CREB is also a transcriptional activator of PGC1α and can therefore also stimulate mitochondrial biogenesis 73 . Finally, increased levels of cGMP induced by caloric restriction and the inhibition of phosphodiest

Mitophagy in most cell types is regulated by a PTEN-induced putative kinase 1 (PINK1)– PARKIN mechanism that tags mitochondria for degradation 109 . PINK1, a kinase that is located in the cytosol, is imported into the mitochondria and then degraded under physiological conditions 110 . As protein import is dependent on the mitochondrial membrane potential, mitochondrial depolarization results in an accumulation of PINK1 on the outer membrane; the PINK1-mediated phosphorylation of certain proteins on the outer membrane mediates recruitment of the E3 ligase, PARKIN 111 – 114 , to the outer membrane. PARKIN ubiquitylates lysine residues in the N-termini of mitochondrial outer membrane proteins, such as MFN1 and MFN2, thereby targeting the mitochondria for degradation by autophagosomes 115 – 119 . Several pathways regulate mitophagy ( FIG. 5 ). Proteins that are important for autophagy, such as ULK1 and ULK2, can mediate mitophagy under different stimuli 120 . For example, when nutrients are sufficient, AMPK is inhibited and mTOR inhibits ULK1, suppressing mitophagy 121 . During nutrient deprivation, AMPK is activated and inhibits mTOR, facilitating ULK1 activation and mitophagy 120 ( FIG. 3 ). Under oxidative stress, AMPK can be activated and inhibit mTOR, again stimulating mitophagy 55 , 121 . A more direct role for AMPK in the activation of mitophagy has also been suggested 122 , whereby AMPK directly phosphorylates MFF on Ser155 and Ser172, triggering fission and, subsequently, mitophagy 123 . However, external stimuli that trigger this pathway are unknown and more research is needed. Other stimuli, such as hypoxia, cause the Ser/Thr protein phosphatase phosphoglycerate mutase family member 5 (PGAM5) to dephosphorylate its substrate, the mitophagy receptor FUN14 domain-containing protein 1 (FUNDC1) 124 . FUNDC1 then interacts with microtubule-associated protein 1 light chain 3 (LC3), which mediates the formation of an autophagic membrane 124 , 125 . Alternatively, hypoxia can induce mitophagy through the actions of BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) and NIP3-like protein X (NIX; also known as BNIP3L) via a mechanism involving HIF1α 126 , 127 . HIF1α can directly induce the transcription of BNIP3 and NIX by binding to the promoter of BNIP3 and by recruiting other co-activator proteins to NIX . NIX and BNIP3 are transmembrane proteins located in the mitochondrial outer membrane and can activate mitophagy by dissipating the mitochondrial membrane potential and interacting with LC3 to deliver mitochondria to the autophagosome 127 – 130 . BNIP3 and NIX are also apoptotic regulators that can induce cell death or autophagy by increasing the production of ROS, by binding to pro-apoptotic proteins of the BCL-2 family, or by inhibiting the GTP-binding protein RHEB, an upstream activator of mTOR 131 – 133 . Previous studies suggest that crosstalk exists between both of the mechanisms that can regulate mitophagy 127 , 134 , 135 , although the mechanisms of this proposed crosstalk are unclear and additional studies are needed to determine the mechanisms that regulate mitophagy in renal disease.

Diabetic nephropathy is the leading cause of end-stage renal disease (ESRD) in the USA 177 , 178 . It is characterized by hyperglycaemia, albuminuria, the accumulation of extracellular matrix proteins, and glomerular and tubular epithelial hypertrophy, as well as a reduced glomerular filtration rate following an initial period of hyperfiltration 179 . Mitochondrial energetics are altered in diabetic nephropathy owing to increased ROS and hyperglycae-mia 180 , both of which induce changes in the ETC that cause a decrease in ATP production and an increase in apoptosis 180 . In line with these observations, increased fission, mitochondrial fragmentation and reduced levels of PGC1α are all observed in the early stages of diabetes mellitus 181 , 182 . Structural changes in mitochondria correlate with the observed changes in mitochondrial energetics 182 . Hyperglycaemia is the main factor that contributes to the development of diabetic nephropathy ( FIG. 7 ). Hyperglycaemia increases the production of NADH and FADH 2 by the TCA cycle, fueling the ETC 183 . ROS released from the ETC can damage mtDNA, hindering the production of mitochondrial proteins 183 . The hyperglycaemic state was originally thought to cause mitochondrial dysfunction by stimulating the development of hyperpolarized mitochondria, which produce more ATP and release higher levels of superoxide from complexes I and III than healthy mitochondria 180 , 184 , 185 . Administration of antioxidants such as vitamin E and vitamin A did not, however, attenuate the complications of patients with diabetes mellitus, suggesting that mitochondrial ROS might not be the primary mediator of mitochondrial dysfunction in diabetic nephropathy 186 . Hyperglycaemia can also increase the level of advanced glycation end products (AGEs), and the activity of the protein kinase C (PKC) and hexosamine pathways, which can contribute to mitochondrial dysfunction 187 . All three events cause deleterious effects that include increased fibrosis, thrombosis, oxidative damage and abnormalities in the vasculature and in blood flow 187 . Hyperglycaemia also stimulates the conversion of glucose to fructose via the polyol pathway in proximal tubules, leading to ATP depletion 188 . A role for endogenous fructose metabolism in the regulation of diabetic nephropathy was suggested by a study showing that deleting the gene that encodes ketohexokinase (KHK; also known as hepatic fructokinase) — the enzyme responsible for the conversion of fructose to fructose-1-phosphate — protected mice from streptozotocin -induced diabetic nephropathy 189 . Proximal tubules are a major site of ketohexokinase expression 188 , 190 and ATP levels were increased and tubular morphology was improved in diabetic Khk −/− mice compared with that of diabetic wild-type mice, suggesting a role for fructose metabolism in the pathogenesis of diabetic nephropathy 189 . Mitochondrial fragmentation has been observed in proximal tubules in the early stages of diabetes mellitus 181 , although the mechanisms that drive changes in mitochondrial dynamics in diabetes are not yet clear. Fission dissipates the mitochondrial membrane potential, decreasing the production of ATP and promoting apoptosis 191 . Several studies have suggested a role for RHO-associated protein kinase 1 (ROCK1) signalling in activating fission in the diabetic kidney 192 . ROCK1 promotes the translocation of DRP1 to the mitochondria and triggers fission by phosphorylating DRP1 (REF. 192 ). Deletion of ROCK1 in mice with streptozotocin-induced diabetes prevents mitochondrial fission, attenuates the increase in ROS production and restores bioenergetic function in the kidney 192 . Patients with diabetes mellitus have reduced levels of the fusion protein MFN2 193 . In line with this finding, kidney-specific overexpression of MFN2 protects rats from streptozotocin-induced diabetic nephropathy 193 . MFN2 overexpression decreased ROS production, decreased kidney volume and attenuated the pathological changes seen in the diabetic kidney 193 . Induced in high glucose 1 (IHG1; also known as THG1L) is another protein that is involved in mitochondrial fusion and has been shown to regulate mitochondrial dynamics and biogenesis in the diabetic kidney 194 . IHG1 can enhance the ability of MFN2 to bind to GTP and interacts directly with MFN2 to mediate fusion 194 . Inhibition of IHG1 reduces ATP production and hinders fusion in vitro 194 . IHG1 also stabilizes PGC1α activation 195 . Reduced levels of PGC1α have also been observed in diabetic rat kidneys 196 . The overexpression of PGC1α in mesangial cells in vitro attenuated the pathophysiological changes induced by hyperglycaemic conditions 196 . The decrease in mitochondrial biogenesis in diabetic rat kidneys is consistent with the translocation of DRP1 to the mitochondrial outer membrane and an increase in mitochondrial fragmentation 196 . The levels of PGC1α mRNA and protein were also reduced in podocytes that were cul

PPARs can regulate cellular metabolism, mitochondrial function, mitochondrial biogenesis, fatty acid oxidation and glucose homeostasis; thus, targeting them could be beneficial for patients with renal disease related to mitochondrial dysfunction. Activation of PPARs can ameliorate ischaemic AKI 207 – 209 . As discussed above, an accumulation of fatty acids and increased ROS production can decrease the efficiency of the ETC. Defects in fatty acid oxidation have been attributed to the downregulation of PPARs during renal ischaemia 18 . Fenofibrate, which is used to treat dyslipidaemia , activates PPARα 210 ( TABLE 1 ). Activation of PPARα leads to activation of lipoprotein lipase, which hydrolyses triglycerides into glycerol and free fatty acids for metabolism 210 . PPARs can also stimulate mitochondrial biogenesis; for example, compounds such as bardoxolone increase the level of PPARG (encoding PPARγ) and NFE2L2 (encoding NRF2) mRNA, leading to mitochondrial biogenesis 211 . However, the use of bardoxolone in clinical trials for patients with type 2 diabetes mellitus and stage 4 CKD showed adverse effects in patients, including an increase in the rate of heart failure events, resulting in termination of the trial 212 . The efficacy of PPAR agonists in animal models suggests these agents could show promise for the treatment of diabetic nephropathy. Treatment of db/db diabetic mice with fenofibrate led to decreased hyperglycaemia and insulin resistance, potentially by correcting glucose homeostasis 213 . Studies have also shown that treatment of diabetic mice with fenofibrate leads to a decrease in fatty acids in the kidney, supporting its potential as a therapeutic for diabetic nephropathy 214 – 216 . These in vivo studies provide evidence that fenofibrate might be suitable for the treatment of patients with diabetic nephropathy. Indeed, fenofibrate decreased dyslipidaemia and albuminuria in patients with type 2 diabetes mellitus and reduced the risk of further cardiovascular events 217 . Taken together, these studies confirm that PPARs have a role in diabetic nephropathy and are a therapeutic target.

A 2014 study described a family of peptides, called Szeto–Schiller peptides (SS peptides), which specifically target cytochrome c activity in the ETC, enhancing its efficiency and increasing ‘state 3 respiration’ — that is, ATP production in the presence of excess substrates and ADP 218 . SS peptides are highly polar, water-soluble tetrapeptides that can cross the blood–brain barrier and specifically target the inner mitochondrial membrane. The SS peptides do not cause mitochondrial depolarization upon entry, making these compounds highly promising for treatment. SS peptides prevent the peroxidation of cardiolipin, a phospholipid that is important for maintaining cristae formation, by cytochrome c 218 . Cytochrome c binds to and oxidizes cardiolipin, disrupting cristae formation and detaching cytochrome c from the inner mitochondrial membrane 219 , 220 . The SS-31 peptide (also known as elamipretide) has been shown, in a variety of animal disease models, especially in AKI, to promote ATP recovery and cristae formation 218 . Pretreatment of rats with SS-31 in vivo maintained cristae formation and prevented mitochondrial swelling of renal tubular epithelial cells 218 . Due to the success in animal models, SS-31 is currently in clinical trials for the treatment of impaired renal function 221 ( TABLE 1 ).