Research progress of autophagy in pathogenesis of diabetes nephropathy

3.1 Mammalian target of rapamycin (mTOR) pathway

mTOR, as an important nutrient energy receptor in the cell and a typical silk/threonine protein kinase, participates in multiple signal pathways and promotes the synthesis and secretion of important substances in the cell by sensing signal molecules such as amino acids, glucose, and growth factors [15]. Generally speaking, mTOR exists in the form of mTOR complex 1 (mTORC1) and mTORC2 complexes. According to the research, mTORC1 can regulate cell growth by integrating the nutrition, hormones, and cytokines of the environment where many cells are located, and play a more important role in autophagy [16,17,18]. Apart from that, autophagy levels can also be regulated by phosphorylating transcription factor EB (TFEB) or inhibiting TFEB nuclear translocation [19,20]. In the case of nutrient deficiency, mTORC1 is inhibited and ULK1 is stimulated by AMPK to promote autophagy [21,22].

When DN occurs, the long-term high glucose environment will enhance mTORC1 activity and inhibit autophagy [23]. According to some studies, mTORC1 is increased in patients with type 1 and type 2 diabetes and in laboratory diabetes animal models [24]. Additionally, the specific enhancement of mTORC1 can cause DN-like pathological damage and clinical features of proteinuria in mice [25]. Therefore, inhibiting the activity of mTORC1 can delay the progression of DN and improve renal function. Rapamycin, as a specific binding agent of mTORC1, can directly act on the FK506 binding protein 12 (FKBP12)-rapamycin binding (FRB) domain of mTOR, inhibit the activity of mTOR and promote autophagy after binding with the intracellular receptor FKBP12 [26]. Currently, the very low protein diet therapy for diabetes patients can inhibit the activity of mTORC1 and enhance the autophagy level of renal tubules in diabetes rats [6]. In addition to the targeted therapy against mTORC1 protein, increasing numbers of studies on autophagy-related signaling pathways including mTORC1 have been conducted in recent years. It was confirmed that adipose stem cells (ADSCs)-derived exosomes (ADSCs-exo) could reduce the expression of Smad1 by enhancing the effect of microRNA-486 (miR-486, a key factor of ADSC) on the 3′-untranslated regions (3′-UTR) of Smad1 (an upstream signal molecule of mTORC1) and increase autophagy flux by inhibiting Smad1/mTORC1 signaling pathway [27]. Meanwhile, curcumin can up-regulate the expression of E-cadherin and autophagy-related protein LC3, down-regulate the expression of phosphorylated mTOR (p-mTOR), p-Akt and PI3K, regulate autophagy flux through PI3K/Akt/mTOR pathway, and increase autophagy vacuoles [28].

3.2 AMPK pathway

AMPK is a nutrient sensing kinase that can be activated by several upstream phosphorylated kinases, including liver kinase B1 (LKB1), calcium/calmodulin dependent kinase kinase β (CaMKKβ), and TGF-β-activated kinase 1 (TAK1) [29,30]. AMPK is mainly regulated by intracellular adenine nucleotide levels (adenosine triphosphate [ATP], adenosine diphosphate [ADP], and adenosine monophosphate [AMP]). When the body is low in energy, the ATP/AMP ratio drops sharply. AMP can bind to AMPK and promote the upstream kinase LKB1 to phosphorylate AMPK at Thr172 [31]. However, AMPK is inhibited when energy is surplus. Numerous studies, both in vivo and in vitro, have revealed that a high sugar diet can lead to decreased AMPK activity in tissues, and the main mechanism is that glycogen binds to the carbohydrate-binding domain (CBD) of AMPK subunits and inhibits conformational changes in AMPKβ, thereby reducing its activity [32]. Unlike mTORC1, AMPK is a potent positive regulator of autophagy, which can regulate autophagy activity through inhibiting mTORC1. Indeed, AMPK can directly phosphorylate autophagy-related protein ULK1 downstream of mTORC1, phosphorylate the mTOR regulatory protein raptor protein or tuberous sclerosis complex 2 (TSC2) to inhibit the activation of mTORC1, and block the inhibition of mTORC1 on ULK1, thereby promoting autophagy [33, 34, 35]. Beyond that, AMPK can not only promote autophagy by phosphorylating PI3K directly, but also promote autophagy by phosphorylating ATG9 or Beclin1 in PI3K [36,37]. In DN, high glucose inhibits the expression of AMPK, reduces the inhibition of mTORC1 by AMPK and inhibits autophagy. Therefore, artificially intervening the content and activity of AMPK in the occurrence of DN can be an effective target for treating DN.

AMPK agonists can directly restore the Thr172 phosphorylation level on the AMPKα subunit and reduce mTORC1 phosphorylation induced by high glucose [35]. Currently, the common AMPK agonists include metformin, resveratrol, etc. Apart from that, metformin can reduce oxidative stress and inhibit autophagy in kidney injury by activating AMPK and SIRT1, and inhibiting forkhead box O1 (FOXO1) [38]. It was demonstrated in 2021 for the first time that metformin could activate mitophagy via the p-AMPK/PTEN-induced kinase 1 (PINK1)/Parkin pathway and improve renal oxidative stress and tubulointerstitial fibrosis in diabetic model mice [39]. Genipin is widely applied in clinical treatment due to its anti-inflammatory, anti-glycating, anti-angiogenic and anti-diabetic pharmacological properties [40]. As confirmed by recent studies, Genipin can up-regulate the expression of AMPK in DN rat model, and inhibit Akt to directly phosphorylate ULK1 to enhance autophagy activity [40]. Besides, Genipin can also use AMPK/SIRT1/nuclear factor-κB (NF-κB) pathway to block autophagy-related oxidative stress and inflammatory response, slow down DN process and improve prognosis [41].

3.3 SIRT1 pathway

SIRT1 (sirtuin-1) is a nicotinamide adenine dinucleotide (NAD⁺)-dependent protein deacetylase, and an intracellular energy sensor regulated by NAD⁺/NADH levels. It increases when energy is deficient and SIRT1 is activated. SIRT1 regulates metabolic homeostasis and autophagy, resistance to apoptosis, and oxidative stress. It acts through deacetylate of histone proteins and transcription factors p53, FOXO, NF-κB, hypoxia inducible factor 1α (HIF-1α) to suppress inflammation and exert renoprotective effects [42]. As verified by the previous studies, the SIRT1 can promote autophagy through deacetylate of autophagy-related proteins ATG5, ATG7, ATG9 and LC3 to, and enhance the gene expression of Bcl-2 19-kDa interacting protein 3 (BNIP3) through deacetylate of FOXO3a [43].

Upon DN, the SIRT1 levels are decreased and autophagy is inhibited in renal tissues. Recently, several studies have confirmed that the artificial means to increase SIRT1 levels and promote autophagy in renal tissues are beneficial to re-establish renal homeostasis and improve renal function. It is demonstrated that dapagliflozin, an oral hypoglycemic agent commonly used in the clinic, can up-regulate SIRT1 and autophagy-related protein Beclin1 and NPHS2 expression levels, enhance renal cell autophagy in rats with DN, and thereby inhibit podocyte injury. Compared with resveratrol, BF175 is a more potent activator of SIRT1 [44]. BF175 ameliorates diabetic glomerular injury in OVE26 mice by protecting podocytes from high glucose induced injury through improvement of mitochondrial function and homeostasis in a SIRT1 dependent manner [44]. In addition to the pharmaceutical means, more and more experiments have proved that some RNAs, such as miRNAs, long non-coding RNAs (lncRNAs), play important roles in regulating autophagy through regulation of SIRT1 expression. MiR-150–5p targets the 3’-UTR of SIRT1, thereby reducing SIRT1 levels in podocytes. Besides, anti-miR-150–5p targets up-regulates SIRT1 and strengthens the interaction between SIRT1 and autophagy-related gene p53 to enhance autophagy and improve renal function through the SIRT1/p53/AMPK pathway [45]. Another recent study found that SRY-Box transcription factor 2 overlapping transcript (SOX2OT), a kind of lncRNA, could promote SIRT1 expression by sponging miR-9, and over-expression of SOX2OT would alleviate high glucose-induced podocyte injury through autophagy induction via the miR-9/SIRT1 axis [46].

4.1 Oxidative stress and autophagy

Oxidative stress is a result of the imbalance between local antioxidant defense caused by excessive production of ROS, which exceeds the antioxidant clearance capacity of the body [47]. Under normal physiological conditions, the body can produce a small amount of ROS, which can regulate vascular tension, cell adhesion, immune response, and other physiological functions. Besides, excessive ROS will induce the release of a large number of cytokines, proinflammatory markers and growth factors, and indirectly lead to tissue and cell damage. When the kidney is chronically in a hyperglycemic state, the accumulation of intracellular glycogen and lipids will cause abnormal intracellular metabolism, such as the activation of glucose advanced glycation, protein kinase C pathway, and nonenzymatic glycosylation pathway. These processes and high levels of non-esterified fatty acids damage the stability of mitochondria, induce the production of many ROS, disrupt the balance of oxidants/antioxidants in the body, induce oxidative stress, and cause cell damage [48,49]. It has been found that ROS can activate protein kinase R-like endoplasmic reticulum kinase (PERK) via the target protein eukaryotic initiation factor 2α (eIF2α) phosphorylation oxidizes ATG4 protease, promote the level of proteolytically mature LC3, prevent mTOR activation, and thus boost the occurrence of autophagy [50]. In the early stage of DN, intracellular accumulated ROS would promote autophagy, and the short-time treatment of podocytes or tubule epithelial cells with high glucose or angiotensin in vitro could increase intracellular ROS levels, which in turn promotes autophagy [51].

Lipoxin A4 (LxA4), as an eicosanoid derivative from polyunsaturated fatty acid metabolism, can help resolve inflammation by reducing partial oxidative stress. A recent study has showed an up-regulation of the nuclear factor E2-related factor 2 (Nrf2)-heme oxygenase-1 (HO-1) pathway in response to LxA4, which may explain the partial renal protective effect of LxA4 by activating intracellular antioxidant defense mechanisms [52]. RAAS blockers have also been demonstrated to significantly decrease the production of oxidant species and thus slow the progression of end-stage renal disease (ESRD) by inhibiting angiotensin II mediated NADPH oxidase activation. It has been verified in several studies that antioxidant therapy with vitamin E and vitamin C can provide some degree of renoprotection in patients with type 2 diabetes, but its effectiveness is uncertain [53]. The effect of probiotic supplementation on glucose and oxidative stress in patients with type 2 diabetes has been confirmed by a recent clinical meta-analysis, indicating that probiotic supplementation has a significant beneficial effect on serum fasting blood sugar (FBS) levels as well as oxidative and antioxidant stress biomarkers. Thus, it is reasonable to assume that the alteration of gut microbiota caused by probiotic supplementation and its subsequent benign progression may protect renal function in patients with diabetes [54].

4.2 Endoplasmic reticulum stress and autophagy

The endoplasmic reticulum, as the main source of autophagic vesicle membranes, is mainly involved in the process of protein synthesis and maturation, as well as the folding and assembly of some proteins [55]. Mitochondrial ROS overproduction, protein glycosylation, and accumulation of misfolded proteins, etc. cause endoplasmic reticulum (ER) stress in times of overnutrition. In diabetes, the accumulation of misfolded proteins aggravated by high glucose and free fatty acids can not only induce endoplasmic reticulum stress and unfolded protein response (UPR) in podocytes and tubular epithelial cells, but also lead to subsequent cell death. ER stress is mainly mediated by activating transcription factor 6 (ATF6), inositol essential protein 1α (IRE1α) and PERK regulate cell autophagy [56]. As confirmed by previous studies, the PERK can phosphorylate downstream eIF2α, which selectively enhances ATF4 mRNA translation, activates autophagy-related target mTORC1 through eIF2α/ATF4 signaling, and enhances the expression of autophagy-related genes ATG4, ATG5, ATG10, thereby promoting autophagy [57, 58, 59]. Molecular chaperones that enhance protein folding can reduce endoplasmic reticulum stress and attenuate diabetic injury, an effect that may be mediated through repair of defective autophagy. Emodin, which is extracted from the rhizomes of several plants, has been widely applied to reduce inflammation, inhibit cell proliferation, and relieve ER stress. As shown by some studies, emodin exerts protective effects on podocytes in DN by alleviating podocyte apoptosis through inhibition of the PERK/eIF2α signaling pathway in vivo and in vitro [60]. Tauroursodeoxycholic acid (TUDCA) is a molecular chaperone, and persistent high glucose induced autophagy defects in podocytes are significantly corrected by treatment with the ER stress inhibitor TUDCA in db/db mice. Besides, TUDCA can prevent podocyte apoptosis induced by AGEs through blocking endoplasmic reticulum stress mediated apoptosis [23]. It is found that treatment with the molecular chaperone phenylbutyrate could also reduce proteinuria, inhibit expression of the endoplasmic reticulum stress markers PERK and glucose regulated protein 78 (GRP78), and decrease phosphorylated c-Junnh NH(2)-terminal kinase (JNK), monocyte chemoattractant protein-1 (MCP-1), and TGF-β1 in streptozotocin (STZ) induced diabetic rats. Taken together, these studies suggest that ER stress, which is induced by high glucose environment in cells, may be a stress adaptive response. By reducing this stress adaptive response, autophagy activity may be restored to achieve renal protection. However, further studies are needed to establish this causal relationship.

4.3 Hypoxia stress and autophagy

Altered osmolality and subsequent pathologic changes in renal resident cells by a persistent high glucose environment could cause renal filtration overload with altered metabolic problems that increase oxygen consumption. The long-term hypoxic environment enables mitochondria to produce a large amount of ROS. It inhibits the degradation of HIF-1 and allows its active form HIF-1 to accumulate in the body, thereby promoting autophagy through multiple pathways and inhibiting the progression of DN. As revealed by studies, the AGEs produced under high glucose environment can up-regulate the expression of HIF-1α through the activation of HIF-1α/pyruvate dehydrogenase kinase 4 (PDK4) signaling enhances autophagy [61,62]. Meanwhile, HIF-1α can activate BNIP3 and Nix, and activated BNIP3L and Nix bind to Bcl-2, dissociate Bcl-2/Beclin1, and release Beclin1 to induce autophagy production [63]. Moreover, HIF-1α can promote ATG2A, ATG14 expression and inhibit mTORC1 expression to promote autophagy [64]. According to the additional studies, HIF-1α/Jumonji domain-containing protein 1 A (JMJD1A) signaling pathway is involved in inflammation and oxidative stress induced by high glucose and hypoxia, and this pathway may serve as a novel target for oxidative stress and inflammation related events in response to diabetic vascular injury [65]. Therefore, HIF-1 is considered as a protective factor for DN, which can maintain the autophagic function of renal cells under a high glucose environment.