In this paper, we analyzed the molecular mechanism of reversal of microalbuminuria in early DN based on a proteomic approach with dapagliflozin. GO and KEGG pathway analysis of the differential proteins confirmed that mitochondrial energy metabolism, humoral immunity, and inflammation play key roles in DN kidney injury. The five screened differential proteins might be used as potential biological markers for DN.
Oxidative stress is closely related to DM and its complications [19, 20] and plays an important role in the development of DN and its progression to end-stage renal disease [21, 22]. Oxidative stress is directly associated with podocyte injury, proteinuria, and tubulointerstitial fibrosis , and is associated with altered renal hemodynamics , and has a synergistic effect. Reactive oxygen species (ROS) concentrations can reflect the level of oxidative stress in vivo. Physiological concentrations of ROS can play a role in signal transduction , redox signaling pathways, and maintain intracellular homeostasis; low to moderate concentrations of ROS are beneficial for cell survival, stem cell function, and lifespan extension [26, 27]; excess ROS damage cells by unbalancing the body's oxidative and antioxidant systems , causing DNA, protein, and lipid damage and acting as signaling molecules in a variety of cellular damage pathways [29–34]. Reducing excess ROS has been shown to slow the progression of various DM complications [35–38]. Therefore, inhibition of ROS production may be beneficial in the treatment of DN. there are many pathways of ROS production in DM patients , mainly during mitochondrial respiration [40, 41]. Excessive ROS production is associated with mitochondrial dysfunction, so ROS is considered a biomarker of mitochondrial dysfunction in DN [42–44]. Reduced mitochondrial ATP production, low levels of mitochondrial membrane potential (MMP), and increased mitochondrial debris are associated with tubular cell injury and apoptosis in DN [45, 46]. Chronic hyperglycemia can also trigger mitochondrial fragmentation and promote mitochondrial dysfunction, as evidenced by elevated mitochondrial ROS production [47–49], increased mitochondrial permeability transition pore (mPTP) opening by pro-apoptotic factor leakage, and caspase-9 apoptotic pathway activation, etc. .
The present study hypothesized that dapagliflozin could restore mitochondrial function and exert anti-oxidative stress effects, as a link between SGLT2i and mitochondrial function has been previously reported [51–53]. The first line of defense against ROS-induced damage is the antioxidant enzyme SOD , of which SOD1 is the key enzyme of DN. SOD1 accounts for 85% of the total SOD activity in most mammalian cells  and is present in the cytoplasm, nucleus, peroxisomes, and mitochondria . It can be used to scavenge superoxide anion radicals in organisms , and the excessive production of superoxide anions leads to the formation of secondary reactive oxygen species such as peroxynitrite and hydroxyl radicals. By activating PI3K/Akt signaling pathway, antioxidant drugs can increase SOD1 expression activity, enhance cellular antioxidant capacity, and mitigate oxidative damage, thus effectively preventing and controlling cellular DNA oxidative damage and mitochondrial dysfunction .
Several animal studies [55, 59, 60] and one study with DN patients  showed that SOD1 expression is downregulated in DN, unfortunately, none of these studies specified the stage of DN in which the subjects were studied. DeRubertis F R et al  showed that SOD1-deficient animal models can accelerate renal pathology. Recently Rodrigues A M et al  found that SOD1 expression was upregulated in an animal model of Wistar rats as an adaptive response to oxidative stress. Supplementation of SOD1 suppressed proteinuria and improved renal lesions , suggesting that the severity of DN could be reduced by upregulating SOD1 activity [64, 65], which is consistent with our findings. Unlike previous studies on SOD1, the present study for the first time specified the DN stage in which the study subjects were in Mogensen stages I-III, and the results showed that SOD1 protein expression was upregulated in the DN group compared with the NC group (log2Fc = 10.0041, p < 0.05), while in the DN-DANA/DN group, dapagliflozin restored or changed the protein level toward healthy control (NC group) levels. Previous studies have differed for the expression levels of SOD1 in DN, and we speculate that the possible reasons are:1) SOD1 is involved in the development of DN and the levels change dynamically throughout DN disease.2) SOD1 is upregulated in response to increased oxidative stress, which is a key cellular defense mechanism against excessive oxidative stress. More studies are still needed to clarify the changes of SOD1 protein expression content in different DN stages.
CXCL12 is a chemokine and tissue repair marker widely expressed in multiple organs, highly active under pathological conditions such as inflammation  and ischemia, and promotes stem cell regeneration and tissue repair. It has been reported  that CXCL12 has anti-oxidative stress effects, and the main receptor of CXCL12 is CXCR4 , and disruption of CXCR4 receptors in mice leads to increased endogenous ROS production, and this study demonstrates for the first time that mitochondrial oxidative stress can be reduced through the CXCR4/CXCL12 axis, suggesting that the CXCR4/CXCL12 pathway in regulating intracellular ROS levels. To date, CXCL12 was always found in mice with six different splice variants , all encoded by the CXCL12 gene. In contrast, only two isoforms, CXCL12α and CXCL12β, were found in humans , with more CXCL12α than CXCL12β. Therefore, we can assume that in the present study it was mainly CXCL12α that played a role. CXCL12α is recognized as a protective mediator of cell and tissue repair in DM and NDM kidney diseases [70, 71]. The results of the present study showed that CXCL12 expression was upregulated in the DN group compared to the NC group (log2Fc = 1.6263, P < 0.05), presumably because the organism is in the early stages of disease at this time with enhanced oxidative stress and foot cell damage, CXCL12 levels were upregulated as a protective mechanism, exerting anti-oxidative stress and reducing microalbuminuria. CXCL12 expression levels were gradually downregulated after dapagliflozin application (log2Fc = 1.8291, p < 0.05), presumably the mechanism of drug action was anti-oxidative stress. The reasons for the differences in the results of different studies may be related to different disease models and being indifferent DN stages, which still need to be further explored.
A recent study  showed that CXCR4 can maintain MMP and mitochondrial function through the JAK2/STAT3 pathway. STAT3 acts mainly as a transcription factor and is translocated into the nucleus after phosphorylation.STAT3 activation mediates the pathogenesis of DN . Downregulation of CXCL12α expression and reduced binding to CXCR4 will inhibit CXCL12α/ CXCR4 signaling and downstream STAT3S727 phosphorylation, impeding STAT3S727 translocation into mitochondria and ultimately leading to increased mitochondrial fragmentation and altered mitochondrial homeostasis. All of the above studies suggest that CXCL12 can be involved in regulating mitochondrial homeostasis directly or indirectly, regulating ROS levels and oxidative stress, etc.
There is an interaction between SOD1 and CXCL12 receptor CXCR4, and CXCL12 stimulation of cells overexpressing SOD1 is followed by a significant increase in Akt activation . Several studies have shown that Akt is involved in the development of DM and its complications, such as the maintenance of normal glomerular endothelial cell function through the PIK3/Akt pathway , protection against high glucose-induced podocyte injury , and association with various cellular processes such as apoptosis, inflammation, and autophagy [77, 78]. In the present study, SOD1 was significantly more upregulated than CXCL12, which stimulates overexpression of SOD1 leading to increased Akt activation, and maybe a mechanism for improving early DN. The effect on SOD1 after application of dapagliflozin was greater than CXCL12, and the degree of SOD1 downregulation was significantly greater than CXCL12, and Akt activation was attenuated. We speculate that the reasons for this may be: 1) the application of dapagliflozin alleviates the renal oxidative stress damage induced by ROS, and the compensatory protection mechanism of the body is gradually weakened; 2) dapagliflozin directly mediates Akt activation through other signaling pathways other than CXCL12/SOD1 pathway.
SLC25A6, a member of the mitochondrial carrier subfamily, is commonly expressed in all tissues  and is a core component of mPTP. mPTP channels are a supramolecular complex of proteins in the outer and inner mitochondrial membranes and intermembrane spaces that are voltage- and Ca2+-dependent . mPTP opening underlies the absence of mitochondrial membrane potential (Δψm) and is involved in the cell apoptotic process . Endoplasmic reticulum Ca2+ homeostasis is a key determinant of ROS levels and mPTP opening rate . Based on the role of SLC25A6 and the results of KEGG enrichment analysis, we found that SLC25A6 is involved in this pathway of antioxidant defense system together with SOD1, which can regulate the expression level and function of mPTP by acting directly or indirectly on mPTP constructed with the participation of SLC25A6 and located upstream of SLC25A6 (see has05022, hsa05016, and hsa05020). In the early stage of DN development accompanied by elevated blood glucose, excessive ROS production leads the body to be under oxidative stress, while SCL25A6 expression is upregulated in the DN/NC group, so we hypothesized that the excessive ROS and upregulated SLC25A6 expression together activate mPTP, causing the kidney foot cells, thylakoid cells, etc. to swell and rupture, and the mitochondria located in the kidney to become dysfunctional or even cell-induced apoptosis. Combined with the previous paper, it is easy to find that SOD1 expression in DN-DAPA/DN group was significantly down-regulated after the application of dapagliflozin, the oxidative stress effect was gradually weakened, ROS production was reduced, SLC25A6 expression also became down-regulated, the degree of mPTP opening was reduced or even shut down, and the opening rate was reduced, which protected the kidney cells from oxidative stress damage. Therefore, we suggest that dapagliflozin exerts anti-oxidative stress and inhibits mitochondrial apoptosis, which is directly mediated through the inhibition of SOD1/SLC25A6 expression in renal cells. Combined with the interaction between the two and the subcellular structure localization, we infer that CXCL12 regulates SOD1/SLC25A6 extracellularly, so dapagliflozin may further inhibit SOD1/SLC25A6 by first acting on CXLC12 extracellularly to downregulate CXCL12 expression, but further experiments are needed to verify this.
S100A13 is a Ca2+ binding protein and belongs to the S100 protein family .S100A13 protein is involved in Ca2+ homeostasis, energy metabolism, inflammation  and interacts with intracellular transcription factors and DNA, activating surfaces including RAGE and toll-like receptor 4, G protein-coupled receptors [85, 86]. It has been demonstrated that the receptor for S100A13 is the receptor for advanced glycosylation end products (RAGE), which is involved in inflammatory processes, plays a central role in acute and chronic inflammatory diseases, and is closely associated with DM complications .S100 proteins act in an autocrine or paracrine manner after binding to RAGE [88, 89], activating RAGE-mediated signaling pathways. Mondola P et al  demonstrated that SOD1 induces Ca2+ increase through intracellular and extracellular-dependent mechanisms and that SOD1, in addition to acting as a scavenger of superoxide radicals, can also induce Ca2+ increase through intracellular and extracellular-dependent mechanisms, further affecting different biological functions.S100A13, as a Ca2+ binding protein, is bound to be somewhat affected by SOD1-induced changes in Ca2+ content.
PPP2R2A is one of the regulatory subunits of PP2A, and Akt is one of the substrates of PP2A, which is closely related to the development of DM and its complications. PPP2R2A inhibits the Akt pathway by inducing p-Akt dephosphorylation  and thus inactivating Akt function. Our data clearly show that PPP2R2A expression activity was upregulated in the DN group compared to the NC group, at which point it was a protective upregulation response due to oxidative stress, while the DN-DAPA group showed downregulation of this protein expression compared to the DN group, suggesting that the application of dapagliflozin may inhibit PPP2R2A activity and thus play a role in the repair of early DN damage.
From the results of GO enrichment analysis, we found that the Cellular Component of differential proteins gradually changed from extracellular space to nuclear lumen, nucleoplasm, and mitochondria-associated protein complexes under the effect of dapagliflozin, and we can confidently speculate that the mechanism of dapagliflozin action is through the action of Mitochondria, and the application of dapagliflozin has led to more differential proteins with phosphatase function in Molecular function, phosphorylation is pivotal for glucose metabolism and energy storage and release, and also plays a key role in signaling pathways . In summary, dapagliflozin acts mainly in mitochondria, conferring differential protein phosphorylation. It may regulate mitochondrial dysfunction and oxidative stress injury, attenuate early renal lesions, and suppress microalbuminuria by engaging in ROS-dependent inhibition of PPP2R2A and CXCL12/S100A13 pathways. Therefore, this signaling pathway may be a target for clinical treatment of DN, and dapagliflozin acts through this pathway, and the specific mechanism needs to be further investigated.