Role of Impaired Nutrient and Oxygen Deprivation Signaling and Deficient Autophagic Flux in Diabetic CKD Development: Implications for Understanding the Effects of Sodium-Glucose Cotransporter 2-Inhibitors

Abstract

Growing evidence indicates that oxidative and endoplasmic reticular stress, which trigger changes in ion channels and inflammatory pathways that may undermine cellular homeostasis and survival, are critical determinants of injury in the diabetic kidney. Cells are normally able to mitigate these cellular stresses by maintaining high levels of autophagy, an intracellular lysosome-dependent degradative pathway that clears the cytoplasm of dysfunctional organelles. However, the capacity for autophagy in both podocytes and renal tubular cells is markedly impaired in type 2 diabetes, and this deficiency contributes importantly to the intensity of renal injury. The primary drivers of autophagy in states of nutrient and oxygen deprivation—sirtuin-1 (SIRT1), AMP-activated protein kinase (AMPK), and hypoxia-inducible factors (HIF-1α and HIF-2α)—can exert renoprotective effects by promoting autophagic flux and by exerting direct effects on sodium transport and inflammasome activation. Type 2 diabetes is characterized by marked suppression of SIRT1 and AMPK, leading to a diminution in autophagic flux in glomerular podocytes and renal tubules and markedly increasing their susceptibility to renal injury. Importantly, because insulin acts to depress autophagic flux, these derangements in nutrient deprivation signaling are not ameliorated by antihyperglycemic drugs that enhance insulin secretion or signaling. Metformin is an established AMPK agonist that can promote autophagy, but its effects on the course of CKD have been demonstrated only in the experimental setting. In contrast, the effects of sodium-glucose cotransporter–2 (SGLT2) inhibitors may be related primarily to enhanced SIRT1 and HIF-2α signaling; this can explain the effects of SGLT2 inhibitors to promote ketonemia and erythrocytosis and potentially underlies their actions to increase autophagy and mute inflammation in the diabetic kidney. These distinctions may contribute importantly to the consistent benefit of SGLT2 inhibitors to slow the deterioration in glomerular function and reduce the risk of ESKD in large-scale randomized clinical trials of patients with type 2 diabetes.

Type 2 diabetes is accompanied by an inexorable decline in renal function, but the pathogenesis of diabetic CKD is not fully understood. Classically, physicians have long believed that hyperglycemia triggers changes in intrarenal hemodynamics that lead to glomerular hyperfiltration and injury.¹ This conceptual framework underpins the use of drugs that inhibit the renin-angiotensin system, which reduce intraglomerular pressures, presumably by acting to reduce efferent arteriolar tone.² Angiotensin receptor blockers and angiotensin-converting enzyme inhibitors have favorable effects on the development and progression of CKD,³ but renal function still deteriorates in patients with diabetes despite the use of these drugs.

Sodium-glucose cotransporter–2 (SGLT2) inhibitors exert striking effects to slow the deterioration of glomerular function and reduce the likelihood of progression to ESKD in patients with diabetes already receiving inhibitors of the renin-angiotensin system.⁴ SGLT2 inhibitors also can lower intraglomerular filtration pressures, which has been attributed to their influence on the proximal reabsorption of sodium.⁵ It has been proposed that this action of SGLT2 inhibitors (via tubuloglomerular feedback) negates the effects of hyperglycemia, producing excessive afferent arteriolar vasodilatation.⁶ However, this hypothesis has been recently disputed,⁷ and, experimentally, knockout of SGLT2 attenuates glomerular hyperfiltration without preventing renal injury.⁸ Importantly, SGLT2 inhibitors exert favorable effects on the course of diabetic CKD at GFR levels that abolish the actions of these drugs on glycosuria and their effects to reduce glomerular filtration pressures.^9,10 These observations have raised important questions concerning the central role of intrarenal hemodynamic events in mediating the effects of SGLT2 inhibitors on the development of diabetic CKD.

In light of this uncertainty, growing evidence suggests that a critical determinant of injury in the diabetic kidney is oxidative and endoplasmic reticulum stress.¹¹ Oxidative stress follows the overproduction of reactive oxygen species that can result from the deleterious effects of hyperglycemia and glucose intermediates on the structural and functional integrity of mitochondria and peroxisomes.^12,13 Endoplasmic reticulum stress may be triggered by the diabetes-related accumulation of intracellular glucose and lipid pools, as well as unfolded proteins that result from glycation.^14,15 Both forms of cellular stress are highly prominent in the diabetic kidney and can lead to inflammasome activation, the production of proinflammatory cytokines, or the stimulation of other injurious pathways that lead to cellular dysfunction and loss, as well as to inflammation and fibrosis.^16,17 These derangements can affect all kidney cell types, and thus may play a role in the development of podocyte effacement, mesangial expansion, tubulointerstitial inflammation and fibrosis, and renal tubular cell injury and atrophy that is characteristic of diabetic CKD.¹⁸

Role of Autophagy in Diabetic CKD

When oxidative and endoplasmic reticulum stress occurs in the kidney (regardless of cause), its intensity and consequences are normally ameliorated by activation of the housekeeping pathway known as autophagy.¹⁹ Autophagy is an intracellular lysosome-dependent degradative pathway, which maintains cellular homeostasis. Typically triggered by states of nutrient deprivation, autophagy involves the identification and disposal of dysfunctional cellular constituents, particularly mitochondria and peroxisomes, which are the major sources of reactive oxygen species.^20,21 The damaged organelles that are targeted for disposal are encircled by double-layered membranes to form autophagosomes. When the autophagosomes fuse with lysosomes, breakdown of the cargo leads to neutralization of injurious effects, and its molecules are recycled for reuse. Importantly, autophagy can be activated selectively to clear cells of accumulated debris, excessive stores of glucose and lipids, unfolded proteins, and dysfunctional subcellular constituents, which contribute to the pathogenesis of disease.²⁰ Tissue-specific overexpression of autophagy genes is sufficient to extend lifespan, indicating that the activation of autophagy underlies the effects of fasting to prolong life in a broad range of animal species.²²

Autophagy is typically activated by energy deprivation, and it is depressed in states of nutrient overabundance. Type 2 diabetes is characterized by excessive glucose and lipid stores,²³ and the intracellular accumulation of glucose and fatty acid intermediates undermines organellar stability and promotes the formation of unfolded proteins.²⁴ The organelles that are the primary targets for injury, the mitochondria and peroxisomes, are the two most important oxygen-consuming cellular constituents and underlie the enormous oxygen demands of the kidney that are driven by the reabsorptive processes of the renal tubules.²⁵ Accordingly, mitochondrial and peroxisomal dysfunction plays an important role in the genesis of CKD, especially in diabetes.^25,26

Cells might be able to mitigate the oxidative and endoplasmic reticulum stresses produced in the diabetic kidney by stimulating autophagic flux, and podocytes and renal tubules normally maintain high levels of autophagy to sustain their structural and functional integrity.^27,28 However, the capacity for autophagy in both podocytes and renal tubular cells is markedly impaired in type 2 diabetes, and this deficiency contributes importantly to determining the intensity of renal injury.^29–31 The diminution of autophagic flux is related to hyperglycemia and to advanced glycation end products and lipids that accumulate because of deficiencies in glucose and fatty acid oxidation^31–33; these metabolic intermediates can, in turn, be cleared if autophagic flux is enhanced.³⁴ The inadequacy of the autophagic response to cellular stress is a hallmark of diabetic CKD.^35,36 Experimentally, the course of nephropathy can be ameliorated if autophagy is augmented^31,37,38; conversely, it is accelerated if the capacity for autophagy is weakened further.^39,40 Clinically, blood and urine biomarkers of autophagic proteins are depressed in the patients with diabetic kidney disease,^41,42 and renal biopsy specimens of patients with insulin resistance exhibit molecular evidence of autophagy suppression.⁴³

Molecular Determinants of Autophagic Flux in the Kidney and Their Derangements in Type 2 Diabetes

Numerous molecular pathways are capable of modulating autophagy in the kidney, but it is noteworthy that the low-energy sensors sirtuin-1 (SIRT1) and AMP-activated protein kinase (AMPK) act as the primary drivers of autophagy in states of nutrient deprivation. SIRT1 is responsive to levels of NAD, serves as a redox rheostat and a primary response to caloric impoverishment, and is a master regulator of glucose homeostasis.^44,45 AMPK discerns the balance between ATP and ADP/AMP in the cytosol; its activation leads to the breakdown of energy stores, thereby promoting the generation of ATP.⁴⁶ Additionally, oxygen deprivation leads to increased expression and activity of the hypoxia-inducible factors HIF-1α and HIF-2α; their downstream effects act to promote the delivery of oxygen and reduce its utilization.⁴⁷ The effects of SIRT1, AMPK, and hypoxia-inducible factors on autophagy are opposed by activation of the PI3K/Akt/mTOR pathway, which is stimulated by energy overabundance and prioritizes cell growth over survival.^48–50

These enzymes and transcription factors control the activity of hundreds of genes that play a critical role in maintaining cellular homeostasis, and they interact in various ways to promote autophagic flux. The interaction of HIF-1α with Beclin 1 promotes autophagosome formation,⁵¹ and AMPK enhances the maturation of autophagosomes and their fusion with lysosomes,⁵² a process that is opposed by signaling through the PI3K/AKt/mTOR pathway.⁵³ Interestingly, SIRT1 and HIF-2α may act primarily to enhance the autophagy of selected cellular constituents. For example, SIRT1 promotes the clearance of damaged mitochondria,⁵⁴ whereas HIF-2α stimulates the degradation of dysfunctional peroxisomes.⁵⁵ Furthermore, whereas AMPK depresses the activity of HIF-1α,⁵⁶ HIF-1α promotes autophagy in a manner that is AMPK-independent.^57,58 In contrast, the actions of SIRT1 and HIF-2α serve to stimulate and reinforce each other.^59,60

Nutrient and Oxygen Deprivation Signaling and Renoprotection

Importantly, SIRT1 and AMPK exert renoprotective effects that are both dependent on and independent of their effects to promote autophagy. SIRT1 and AMPK are important inducers of autophagic flux in the kidney, and this augmentation is accompanied by a striking amelioration of glomerular and tubular injury in a variety of models of renal stress, including those related to diabetes, aging, ischemia, and drug-related nephrotoxicity.^61–64 Furthermore, these nutrient deprivation transcription factors can inhibit the generation of reactive oxygen species and activation of the inflammasome in ways that are independent of their autophagy-promoting actions.⁶⁵ Both SIRT1 and AMPK act directly to maintain mitochondrial homeostasis, preserve peroxisome integrity and functionality, and enhance the activity of antioxidant mechanisms.^66,67 Additionally, both SIRT1 and AMPK interact directly with a key subunit of NFκB to inhibit its actions, thereby attenuating activation of the NLRP3 inflammasome and muting inflammation-mediated cellular injury.^68,69 Conversely, signaling through the P13K/AKT/mTOR pathway can impair autophagic flux in the kidney.⁷⁰ The pathway also reduces antioxidant activity and promotes oxidative stress and can interact directly with NFκB to stimulate proinflammatory pathways and cell death.^71–73

Derangements in Nutrient and Oxygen Deprivation Signaling in Diabetic CKD

Type 2 diabetes is characterized by marked suppression of the expression and activity of both SIRT1 and AMPK simultaneously with hyperactivation of mTOR, thereby leading to a striking diminution in autophagic flux in both glomerular podocytes and renal tubules and markedly increasing their susceptibility to renal injury (Figure 1).^28–33,74 Hyperglycemia inhibits phosphorylation of the catalytic isoform AMPK-α,⁷⁵ and inhibition and activation of the noncatalytic subunit AMPK-β1 lead to worsening and improvement in the development of diabetic CKD, respectively.^76,77 Similarly, high levels of environmental glucose act to suppress SIRT1 signaling in podocytes and renal tubules,^78,79 but SIRT1 activation mitigates glycemia-related renal dysfunction and injury.^80–82 Conversely, Akt/mTOR signaling is hyperactivated in the diabetic kidney and can trigger proinflammatory pathways and promote renal injury^43,71,83,84; inhibiting mTOR with rapamycin can ameliorate the development of diabetic CKD.^85,86 All of these derangements—acting in concert—reduce the kidney’s ability to maintain cellular homeostasis and health in states of energy overabundance; however, the loss of SIRT1 function seems to be particularly important.⁸⁷ Several epidemiologic studies have shown that polymorphisms in the gene encoding SIRT1 increase the risk of CKD in patients with type 2 diabetes.^88–90 Additionally, serum levels of SIRT1 are decreased in these patients, and this decline parallels the development of albuminuria.⁹¹

Figure 1.

Proposed mechanisms by which type 2 diabetes can lead to cellular stress and CKD. AMPK, AMP-activated protein kinase.

The role of hypoxia-inducible factors HIF-1α and HIF-2α in autophagy and the pathogenesis of diabetic CKD is complex. The kidney’s enormous oxygen requirements are heightened by type 2 diabetes, because renal oxygen consumption is driven by proximal tubular sodium reabsorption,⁹² which is markedly increased in the diabetic kidney.⁹³ This excessive consumption may explain the renal hypoxia that has been seen in diabetes, both experimentally and clinically,^94,95 and this, in turn, may activate HIF-1α signaling. Diabetes can also lead to activation of HIF-1α by a direct effect of hyperglycemia and advanced glycation end products on HIF-1α transcription.^96,97 Regardless of its genesis, the increased activity of HIF-1α has been proposed as a cause of cellular dysfunction and activation of proinflammatory pathways in podocytes, mesangial cells, and renal tubular epithelial cells,^98,99 thus potentially implicating HIF-1α in the pathogenesis of glomerulosclerosis and CKD.¹⁰⁰ However, both HIF-1α and HIF-2α act to promote autophagic flux and thus attenuate oxidative stress and ischemia- and diabetes-related renal injury^57,101–103; HIF-1α promotes the clearance of damaged mitochondria,⁴⁷ whereas HIF-2α enhances the lysosome-mediated removal of dysfunctional peroxisomes.⁵⁵ These actions may explain why pharmacologic enhancement of hypoxia-inducible factor signaling can prevent renal injury and the development of diabetic and nondiabetic CKD, especially when the disease process is well established.^104–108 Therefore, although the available evidence is conflicting, it is possible that the intensity of the HIF response in the diabetic kidney modulates the development of renal injury in an isoform- and time-dependent manner.

Effects of Nutrient Deprivation Signaling on Renal Sodium Transport

Nutrient deprivation signaling not only influences podocyte and renal tubular health, but it also modulates transmembrane sodium transport in the kidney. In general, sodium movement into cells is increased by transporters than enhance sodium influx (e.g., the sodium-hydrogen exchanger [NHE] and the epithelial sodium channel [ENaC]), and it is decreased by transporters that promote sodium egress out of cells (e.g., sodium-potassium ATPase). An imbalance in these transporters can lead to a nonphysiologic increase in intracellular sodium; this impairs the ability of mitochondria to generate ATP and deploy antioxidant defense mechanisms,^109–111 thus contributing to oxidative stress and cell death. Consequently, overactivity of HE or ENaC or suppression of sodium-potassium ATPase can cause increases in intracellular sodium that can trigger cellular demise.^112–116 Oxidative stress in the kidney stimulates transporters driving sodium influx (e.g., NHE or ENaC), while inhibiting those involved in sodium efflux (e.g., sodium-potassium ATPase).^117–119

Importantly, type 2 diabetes is characterized by activation of both NHE and ENaC and decreased activity of sodium-potassium ATPase^120–122; the resulting increase in intracellular sodium leads to mitochondrial stress and cell loss, thus contributing to the development of CKD. Furthermore, if the sodium influx mechanisms (e.g., NHE3) are activated in the proximal renal tubules, the resulting enhancement of sodium reabsorption can lead to a reduction in sodium delivery to the macula densa. This, through tubuloglomerular feedback facilitated by the ENaC, could cause afferent arteriolar vasodilatation and glomerular hyperfiltration, with its attendant risks of glomerular injury.^93,123 In experimental models, inhibition of sodium influx (i.e., NHE and ENaC) and/or enhancement of sodium efflux (i.e., sodium-potassium ATPase) attenuates the adverse changes in the kidneys that are produced by nutrient overabundance.^124–126

It is therefore noteworthy that nutrient deprivation signaling and the P13K/Akt/mTOR pathway modulate the function of ion channels in the kidney.^127,128 Both SIRT1 and AMPK downregulate ENaC and other mechanisms of sodium reabsorption in the renal tubules, but they increase the activity of sodium-potassium ATPase to promote sodium efflux.^129–133 Conversely, mTOR promotes sodium influx through activation of ENaC.¹³⁴ Overall, there is an inverse relationship between autophagic activity and the activation of mechanisms that promote the entry of sodium into cells. Increased autophagic activity leads to enhanced degradation of ENaC and decreased expression of NHE3,¹³⁵ whereas increased NHE3 activity impairs autophagy.¹³⁶ Conversely, there is a direct relationship between the activation of nutrient and oxygen deprivation signaling and the upregulation of mechanisms that enhance sodium efflux (e.g., sodium-potassium ATPase)—that is, activation of AMPK/SIRT1 stimulates sodium-potassium ATPase,^133,137 and activation of sodium-potassium ATPase promotes autophagic flux.¹³⁸ Additionally, inhibition of sodium-potassium ATPase is accompanied by downregulation of HIF-1α,^139–141 and conversely, activation of HIF-1α/HIF-2α leads to inhibition of sodium-hydrogen exchange.¹⁴²

Therefore, the totality of evidence suggests that there is a close linkage between enhanced nutrient and oxygen deprivation signaling (AMPK/SIRT/HIF-1α/HIF-2α) and the attenuation of sodium transport mechanisms that leads to increases in intracellular sodium that can undermine mitochondrial stability. Furthermore, by inhibiting sodium influx in the proximal tubule, activation of nutrient deprivation signaling may also ameliorate the sodium hyper-reabsorption and the abnormalities in tubuloglomerular feedback that are seen in diabetes and that have been implicated in the development of CKD.^93,143,144

Effects of Antihyperglycemic Drugs on Nutrient and Oxygen Deprivation Signaling and on Autophagic Flux in the Diabetic Kidney

Because hyperglycemia and advanced glycation end products can injure the kidney, long-term treatment with antihyperglycemic agents might be expected to slow CKD progression. In randomized, controlled clinical trials, glycemic control in patients with type 1 diabetes has prevented the development of proteinuria,¹⁴⁵ and prolonged treatment with glucose-lowering drugs, combined with intensive treatment of other risk factors, has decreased the likelihood of progression to ESKD in type 2 diabetes.¹⁴⁶

Effects of Insulin-Signaling Antihyperglycemic Drugs on Autophagic Flux and the Clinical Course of Diabetic CKD

Despite these favorable effects, the benefits of antihyperglycemic drugs per se on the course of CKD in type 2 diabetes remain unclear. In the U.K. Prospective Diabetes Study 33 trial, patients receiving insulin or sulfonylureas did not show amelioration of CKD (as reflected by proteinuria or doubling of serum creatinine), despite the drugs’ blood glucose–lowering effects.¹⁴⁷ Furthermore, in the Action in Diabetes and Vascular Disease: Preterax and Diamicron Modified Release Controlled Evaluation (ADVANCE) trial, the Action to Control Cardiovascular Risk in Diabetes trial (ACCORD), and the Veterans Affairs Diabetes Trial (VADT), patients who received intensive glycemic control with insulin, sulfonylureas, and thiazolidinediones showed little or no reduction in proteinuria and no preservation of renal function (as assessed by serum creatinine) compared with patients who received less aggressive glucose lowering.^148–150 Although the ADVANCE trial found a reduction in ESKD,¹⁴⁹ this finding was on the basis of few events and the other two trials observed no such reduction. Long-term therapy with glucagon-like peptide–1 receptor agonists and dipeptidyl peptidase–4 inhibitors has yielded little or no slowing of the rate of decline in glomerular function and no reduction in the risk of major adverse renal outcomes, despite achieving a decrease in albuminuria.^151–154 Therefore, on the basis of the totality of evidence, drugs that promote insulin secretion or signaling have not demonstrated consistently favorable effects on the development of diabetic CKD, even when glucose lowering has been maintained for many years.

Are these disappointing results related to a lack of a benefit of insulin secreatogues or sensitizers on autophagic flux? Any lowering of blood glucose might be expected to increase SIRT1/AMPK signaling, and both liraglutide and pioglitazone appear to promote autophagy in isolated renal tubular cells or in experimental nondiabetic CKD.^155,156 However, in patients with type 2 diabetes, these drugs increase circulating levels of insulin and insulin stimulates the PI3K/Akt/mTOR pathway in the kidney, an action that is preserved in states of insulin resistance.^157,158 Such an effect is accompanied by the suppression of autophagy genes as well as an enhancement of sodium reabsorption.¹⁵⁹ Glucagon-like peptide–1 signaling can impair autophagic flux under conditions of nutrient overabundance.¹⁶⁰ Additionally, dipeptidyl peptidase–4 inhibitors potentiate the actions of the stem cell chemokine stromal cell–derived factor 1, as well as signaling through the latter’s receptor, CXCR4,^161,162 and CXCR4 agonism depresses autophagic flux.¹⁶³ For all of these reasons, antihyperglycemic drugs that promote insulin signaling may be unable to ameliorate the deficient autophagic flux in the diabetic kidney, and they thus may be limited in their effects to favorably influence the course of diabetic CKD.

Effects of Metformin on Nutrient Deprivation Signaling and Autophagic Flux in the Diabetic Kidney

Although metformin is routinely used as first-line therapy to achieve glycemic control in type 2 diabetes, its benefit on the clinical course of diabetic CKD has not been established.^164,165 The drug does not ameliorate (and it may exacerbate) albuminuria,^166,167 and it has not been shown to reduce the risk of serious adverse renal events. In patients at risk of developing diabetes, metformin has not reduced the risk of developing CKD, even though it decreases the likelihood of the development of diabetes.¹⁶⁸

In contrast to this lack of evidence for a beneficial effect of metformin on diabetic CKD from trials or observational studies in the clinical setting, the drug has been shown to ameliorate the development of CKD in animal models of diabetes. This benefit has been attributed to the drug’s actions to mitigate oxidative stress, inflammation, podocyte and tubular injury, and fibrosis.^169–173 The mechanisms that underlie these potential benefits of metformin are unknown, but the drug is an established agonist of AMPK. Although metformin has also been reported to stimulate SIRT1 and inhibit PI3K/Akt/mTOR signaling,^174,175 it is doubtful that these secondary effects mediate the drug’s actions. Metformin inhibits gluconeogenesis and ketogenesis, as well as mitochondrial respiratory-chain complex 1,^176,177 and such actions are inconsistent with enhanced signaling through SIRT1.^178,179 Therefore, it is not surprising that numerous studies have shown that it is metformin’s effect on AMPK signaling that underlies its ability to promote autophagic flux and alleviate cellular stress in different renal cell types.^180,181 The effect on AMPK also appears to explain the drug’s ability to ameliorate the development of the inflammation and fibrosis that characterizes experimental kidney disease in states of nutrient overabundance.¹⁸² However, it is not clear whether AMPK signaling alone can ameliorate the course of diabetic CKD in the clinical setting.^164,165

Effects of SGLT2 Inhibitors on Nutrient Deprivation Signaling and Autophagic Flux in the Diabetic Kidney

The most persuasive evidence for a benefit of any glucose-lowering drug on the development and clinical course of diabetic CKD exists for the SGLT2 inhibitors. Large-scale randomized, placebo-controlled trials have demonstrated the ability of SGLT2 inhibitors to reduce diabetes-related deterioration in glomerular function, as well as to decrease the risk of serious adverse renal events and ESKD.⁴ The magnitude of this benefit is clinically impressive and evident after only 1–3 years of treatment; this benefit is not related to these drugs’ effects on glycemic control.

Although SGLT2 inhibitors can lower intraglomerular filtration pressures,⁵ their actions on arteriolar and efferent arteriolar tone have been debated and disputed.^6,7 Importantly, interference with the actions of SGLT2 can attenuate glomerular hyperfiltration without attenuating renal injury,⁸ and SGLT2 inhibitors’ renoprotective effects are apparent even when there is sufficient glomerular dysfunction to abolish the actions of these drugs on glycosuria and on intrarenal hemodynamics.^9,10

SGLT2 is a biologic indicator of nutrient excess, and thus there is an inverse relationship between SGLT2 activity and the expression of SIRT1 and AMPK in the kidney. High levels of renal tubular glucose promote the expression of SGLT2 but reduce the expression of SIRT1^79,183; increases in SGLT2 promote oxidative stress in the kidney.¹⁸⁴ At the same time, hypoxia causes simultaneous downregulation of SGLT2 but upregulation of AMPK.⁵⁷ Most interestingly, experimental knockout of SGLT2 in the renal proximal tubule appears to promote autophagic flux.¹⁸⁵ These observations suggest that proximal tubular SGLT2 may be an entry point for therapeutic interventions that aim to stimulate nutrient deprivation signaling, i.e., SIRT1 and AMPK. In light of this conceptual framework, it is noteworthy that inhibition of SGLT2 induces a fasting-like transcriptional paradigm¹⁸⁶ that is characterized by loss of calories in the urine, shrinkage of adipose tissue depots, and promotion of gluconeogenesis and ketogenesis (the classic biomarkers of starvation). Additionally, SGLT2 inhibitors appear to induce a state of hypoxia mimicry (akin to that produced by cobalt¹⁸⁷) that is characterized by increased erythropoietin and erythrocytosis.¹⁸⁸

Importantly, the ketonemia and erythrocytosis produced by SGLT2 inhibitors may provide important molecular clues concerning the mechanisms that may underlie the renoprotective effects of SGLT2 inhibitors. A critical determinant of increased ketone body production appears to be the activation of SIRT1 signaling, because SIRT1 promotes both gluconeogenesis and fatty acid oxidation, which are the two most important pathways for ketogenesis.^45,189,190 Additionally, SIRT1 signaling activates the rate-limiting step in ketone body synthesis.¹⁹¹ At the same time, the principal driver of erythropoietin synthesis and erythrocytosis is the activation of the hypoxia-inducible factors HIF-1α and HIF-2α, and HIF-2α (the isoform most closely linked to red blood cell production¹⁹²) is activated by SIRT1.^59,60

In light of their effects to promote ketogenesis and erythropoiesis (the two hallmarks of SIRT1 signaling), it is not surprising that studies show that SGLT2 inhibitors enhance the activation of SIRT1 in diverse tissues, including the kidney,^{79,183,193–195} and there is evidence that SGLT2 inhibitors may directly interact with SIRT1.¹⁹³ The combined activation of SIRT1/HIF-2α may underlie the ability of SGLT2 inhibitors to promote autophagic flux (Figure 2).^{55,185,196–198} Additionally, SGLT2 inhibitors may influence HIF-1α in a manner that favorably affects the course of CKD.^199,200 The hypothesis that SGLT2 inhibitors exert their effects to slow diabetic CKD progression primarily by modulating signaling through low energy–triggered enzymes and transcription factors (SIRT1 and hypoxia-inducible factors) and by stimulating autophagy is worthy of further study.

Figure 2.

Mechanisms by which antihyperglycemic drugs may influence nutrient and oxygen deprivation signaling, autophagic flux and the course of diabetic nephropathy. AMPK, AMP-activated protein kinase; SGLT2, sodium-glucose cotransporter 2.

Similarities and Differences in the Effects of Metformin and SGLT2 Inhibitors on Nutrient Deprivation Signaling in the Diabetic Kidney

Importantly, the effects of SGLT2 inhibitors on nutrient deprivation signaling and autophagy can be distinguished from those of metformin, which acts primarily as an AMPK agonist and does not induce a state of fasting or hypoxia mimicry. Although canagliflozin can activate AMPK directly, empagliflozin and dapagliflozin may not exert such an effect^201,202; nevertheless, an action of these SGLT2 inhibitors to promote SIRT1 signaling may lead indirectly to stimulation of AMPK, at least to a modest degree.^196,203 These observations raise the possibility that AMPK activation may contribute to the renoprotective benefits of SGLT2 inhibitors.²⁰⁴ The findings of the Empagliflozin Cardiovascular Outcome Event Trial in Type 2 Diabetes Mellitus Patients–Removing Excess Glucose (EMPA-REG OUTCOME) trial support this possibility, showing that metformin partially attenuated empagliflozin’s beneficial effects on the clinical course of nephropathy (53% risk reduction in metformin nonusers versus 32% risk reduction in metformin users, interaction P=0.01).²⁰⁵ It should be noted, however, that patients who were receiving metformin still exhibited a substantial benefit from SGLT2 inhibitors, suggesting that the major renoprotective effect of empagliflozin is not mediated through AMPK.

The effects of SGLT2 inhibitors and metformin on nutrient and oxygen deprivation signaling can be distinguished in other ways. Ketogenesis (a consequence of SIRT1 activation^45,189,190) is seen with SGLT2 inhibitors and not with metformin, presumably because AMPK activation inhibits gluconeogenesis.^176,206 Furthermore, SGLT2 inhibitors can interfere with sodium transport mechanisms (e.g., NHE3),²⁰⁷ and they cause a decrease in intracellular sodium, at least in cardiomyocytes.²⁰⁸ These effects are not seen with metformin, which does not reduce cytosolic sodium²⁰⁹ and has minimal effects on sodium reabsorption, even in salt-sensitive states.²¹⁰ Finally, SGLT2 inhibitors promote erythropoietin synthesis and erythrocytosis,^211,212 whereas metformin use is accompanied by a decrease in hematocrit in clinical trials.²¹³ The latter effect is likely explained by an action of metformin (and other AMPK agonists) to inhibit HIF-1α.^56,214–216 In contrast, the effects of SGLT2 inhibitors on HIF-1α remain uncertain, with studies reporting that these drugs decrease HIF-1α activity in the diabetic kidney and increase its activity in nondiabetic renal injury.^119,200 In light of this uncertainty about the effect of SGLT2 inhibitors on HIF-1α, the erythrocytosis that routinely accompanies use of SGLT2 inhibitors in the clinical setting may be more readily explained by activation of HIF-2α as a result of enhanced SIRT1 signaling.^59,60 In statistical mediation analyses, the intensity of erythrocytosis has predicted the reduction in the risk of heart failure events with SGLT2 inhibitors,^211,212 but the strength of any association between erythrocytosis and the lowering of the risk of adverse renal events in clinical trials of these drugs has yet to be explored.

Conclusions

The principal mechanisms driving renal injury in type 2 diabetes appear to be an increase in oxidative and endoplasmic reticulum stress, exacerbated by an impairment of the kidney’s autophagic capacity to neutralize these injurious processes and their adverse effects on renal health. This deficiency is related to an effect of diabetes to depress nutrient deprivation signaling (SIRT1 and AMPK) in both podocytes and renal tubular cells—and, conceivably, diabetes may also impair the adaptive and enhance the maladaptive actions of hypoxia-inducible factors. Interestingly, these derangements are not effectively ameliorated by antihyperglycemic drugs that enhance insulin secretion or signaling. In striking contrast, however, both metformin and SGLT2 inhibitors are poised to augment autophagic flux and thereby mitigate oxidative stress and inflammation in the renal parenchyma.

Yet, these two classes of drugs differ meaningfully in their effects on nutrient deprivation signaling. Metformin acts primarily as an AMPK agonist, whereas the effects of SGLT2 inhibitors may be best explained by postulating that they induce a fasting- and hypoxia-like transcriptional paradigm that manifests primarily by enhanced SIRT1/HIF-2α signaling. Activation of these transcription factors may explain the actions of SGLT2 inhibitors to promote ketogenesis and erythrocytosis in the clinical setting, effects not seen when patients are treated with metformin. Interestingly, other drugs that promote SIRT1 (e.g., resveratrol) reduce oxidative stress, promote autophagy, and ameliorate renal abnormalities in experimental diabetes.^217,218 Drugs that induce autophagy by activation of HIFs (such as cobalt) can also induce autophagy²¹⁹ and prevent the development of diabetic CKD.¹⁰⁵ Therefore, although the mechanisms of action of metformin and SGLT2 inhibitors may overlap, SGLT2 inhibitors exert molecular effects and clinical benefits on the course of CKD in type 2 diabetes that are distinct from other antihyperglycemic agents.

Disclosures

Dr. Packer has recently consulted for Abbvie, Actavis, Akcea, Amgen, AstraZeneca, Boehringer Ingelheim, Cardiorentis, Daiichi Sankyo, Johnson & Johnson, NovoNordisk, Pfizer, Relypsa, Sanofi, Synthetic Biologics, and Theravance.

Funding

None.