Introduction
Diabetes mellitus (DM) is a common chronic metabolic disease characterized by persistent hyperglycemia caused by absolute or relative insulin deficiency and insulin resistance. The estimated number of patients with DM is projected to increase to 700 million by 2045 worldwide [
1]. In addition, DM is the leading cause of chronic kidney disease (CKD) and end-stage renal disease (ESRD). Approximately 40% of patients with DM gradually progress to CKD and even develop ESRD [
2]. Unfortunately, the treatment of DKD remains an unresolved challenge worldwide. To date, strict glycaemic and blood pressure control, especially with the use of angiotensin-converting enzyme inhibitors and angiotensin II receptor blockers, has been shown to delay the progression of DKD [
3,
4]. However, the benefit of these interventions to DKD outcomes is limited [
5]. Therefore, developing new treatment strategies to delay the progression of DKD is necessary.
The pathogenesis of DKD is multifactorial and still unclear. Accumulating evidence elucidates that various cellular stress responses triggered by metabolic abnormalities contribute to the pathophysiological mechanism of DKD [
6]. The upregulation of sodium-glucose cotransporter 2 (SGLT2), when appearing in diabetes, leads to high transmembrane transport in the proximal tubule and generates a dramatic energy requirement in the renal cortex [
7]. Meanwhile, metabolic reprogramming characterized by tricarboxylic acid cycle (TCA cycle) inhibition and glycolysis enhancement occurs in the renal cortex [
8]. This phenomenon of mitochondrial oxidative phosphorylation activity mismatched with increased glycolytic flux results in the accumulation of intermediates from glucose metabolism and triggers multiple pathogenic signaling pathways, thereby inducing oxidative stress, reductive stress, and various cellular stress responses mediated by energy insufficiency [
9,
10]. Moreover, insulin resistance shifts the pattern of renal biofuel utilization from glucose-mediated aerobic metabolism to fatty acid β-oxidation and amino acid metabolism [
7]. Reversing these metabolic abnormalities has demonstrated encouraging results in improving the outcomes of DKD. For example, inhibited KIM-1-mediated fatty acid uptake in renal tubular epithelial cells can block a series of kidney injury-related events triggered by abnormal lipid metabolism in DKD [
11]; similarly, the reversal of abnormal glycolysis and lipid metabolism in DKD was protective against the pathophysiology of DKD [
8].
SGLT2 inhibitors originally developed for treating type 2 Diabetes mellitus (T2DM) are clinically very effective in halting the progression of DKD [
12‐
14]. Recent studies have demonstrated that SGLT2 inhibitors exert both direct or indirect protective effects on the cardiovascular and renal systems in T2DM, which are independent of their glucose control effects but related to their effects on blood pressure control, glomerular hemodynamic amelioration, RAAS regulation, and anti-inflammation and in reducing glucotoxicity, lipotoxicity, and uric acid. Furthermore, SGLT2 inhibitors downregulate hepcidin and promote erythropoiesis [
15,
16], thereby alleviating renal hypoxia by improving the circulating oxygen supply [
7]. Interestingly, a recent study has demonstrated that dapagliflozin prevents the high glucose-induced metabolic transition shift from lipid oxidation to glycolysis in renal tubular cells by inhibiting HIF-1α [
17]. In contrast, empagliflozin inhibit the over-activated nutrient-sensing signaling pathway by increasing endogenous ketone body production, thus improving energy utilization and reducing renal oxidative stress [
18]. This suggests that the SGLT2 inhibitor effectively corrects metabolic disorders in DKD. However, the mechanisms behind the renoprotective effect of SGLT2 inhibitors are unclear.
This study was aimed to investigate the metabolic effects of SGLT2 inhibitors in DKD via liquid chromatography with tandem mass spectrometry (LC–MS/MS)-based metabolomic and proteomic analyzes of serum and the kidneys, and to provide a deeper understanding of the renoprotective mechanism of SGLT2 inhibitors.
Discussion
In this work, we observed that Empa treatment effectively alleviates the renal pathological changes caused by T2DM; simultaneously, we characterized the specific feature of metabolic reprogramming that occurs in kidney and serum in the mice with DKD via multi-omics analysis and identified five metabolic pathways related to Empa treatment, which involve in renal purine metabolism (local purinergic signaling); pyrimidine metabolism (mitochondrial function); tryptophan metabolism (reductive stress); nicotinate and nicotinamide metabolism (reductive stress) and serum glycine, serine and threonine metabolism (nephrotoxicity). Our results suggest that EMPA treatment improves renal function and morphology by regulating renal metabolic reprogramming in mice with DKD.
In physiological conditions, the filtered amino acids are almost reabsorbed by tubular epithelial cells. Furthermore, the kidney is a crucial organ that participates in the biosynthesis and catabolism of multiple amino acids and their derivatives [
37]. A previous study found that patients with DKD were more predisposed to develop amino acid metabolic derangements than patients with DM or healthy people [
38]. In addition, a recent study confirmed that dapagliflozin intervention could reverse the abnormal expression of multiple amino acid transporters and amino acid degrading enzymes in the renal cortex of diabetic mice [
39]. In this study, amino acids and their derivatives had the highest proportion of metabolites before and after EMPA treatment, whether in the serum or kidney. Among them,
N-Acetylcysteine is the precursor to glutathione in vivo, which has a potent effect of scavenging oxygen free radicals and antioxidants by providing sulfhydryl to increase the synthesis of endogenous glutathione and thus reducing the oxidative damage caused by mitochondrial dysfunction [
40]. A recent study confirmed that Canagliflozin could reverse kidney oxidation damage in isoproterenol-induced mice by elevating the concentrations of endogenous glutathione [
41]. In addition, the expression of most dipeptides in the kidney was considerably different after EMPA treatment. Particularly, Val-Tyr (downregulation to upregulation, FC = from 0.24 to 6.94), known as Ang-(3–4), is the shortest peptide segment of local angiotensin (Ang) I and Ang II derivatives, which can bind to AT2R to activate Ca
2+-ATPase in renal tubular epithelial cells via the cAMP–PKA pathway and antagonist Ang II-mediated Na
+ reabsorption and affects the downstream signal for RAAS activity to promote urinary sodium excretion and vasorelaxant effect, thereby declining blood pressure [
42‐
44]. In this study, Val-Tyr expression increased remarkably after EMPA intervention. According to previous studies [
45], EMPA can promote sodium excretion and decrease blood pressure. Moreover, in a previous study, EMPA treatment inhibited RAAS overactivation in Apo
−/− mice but did not influence the expression of Ang II [
45]. Therefore, we speculate that EMPA serves as an antagonist of RAAS by improving the metabolism of some dipeptides or stimulating their synthesis.
Tryptophan and its metabolites are precursors to various microbial biosynthetic products and metabolites of the host [
46]. The following three pathways are currently associated with tryptophan catabolism: (1) serotonin (5-hydroxytryptamine) metabolism, (2) the indole pathway and (3) the kynurenine (KYN) pathway [
47]. Approximately 90% of 5-HT in the body is metabolized by gut bacteria, with the most typical bacteria being
Clostridium sporogenes belonging to the phylum Firmicutes [
46]. In addition,
Clostridium sporogenes can also utilize tryptophan to produce indoleacetic acid (IAA) and indole-3-propionic acid (IPA) via the indole metabolic pathway [
46]. A recent study demonstrated that the proportion of gut bacteria belonging to Firmicutes in db/db mice declined after dapagliflozin intervention [
48], which partly explains the downregulation of intermediates in both serotonin (5-hydroxy-
l-tryptophan) and indole (indole-3-acetaldehyde) pathways after EMPA treatment in this study. High expression of 5-HT and its end product metabolite (5-hydroxyindoleacetic acid) in the serum is strongly associated with the pathogenesis of renal dysfunction in DM [
49], whereas 5-HTR antagonists exert renoprotective effects in DKD [
50], suggesting that EMPA plays a renoprotective role by regulating the metabolism of gut bacteria. Most of the tryptophan is degraded via the KYN pathway in the cycle.
Tryptophan, which acts on a series of enzymes, eventually produces acetyl-CoA through the KYN pathway, which completely decomposes to CO
2 and water after entering the TCA cycle (Fig.
7). Some intermediate metabolites are produced with this process, especially quinolinic acid, which mediates most of the de novo synthesis of NAD+. NAD+ in the kidney is majorly derived from de novo synthesis of NAD+ mediated by tryptophan metabolism [
51,
52]. However, a previous study found that P5P phosphatase (a cofactor for KYNase and a key enzyme regulating the 3-hydroxykynurenine metabolism downstream) is inhibited owing to chronic inflammation in DKD, which blocks tryptophan metabolism to produce NAD+ through the KYN pathway [
53]. This finding is consistent with the accumulation of KYN and its metabolites (3-hydroxykynurenine [3-HKYN] and kynurenic acid [KYNA]) observed in chronic kidney disease such as DKD [
47]. The accumulation of KYN and its metabolites mediate and enhance oxidative stress, immune activation and inflammatory reaction to exacerbate renal damage [
47]. In this study, the expression of acetyl CoA (end product of the KYN pathway) and nicotinic acid mononucleotide (NAMN) (downstream metabolites of quinolinic acid) was upregulated in the kidney of db/db mice after EMPA intervention, indicating that EMPA may reverse renal dysfunction by promoting KYN metabolism to ameliorate NAD+ levels.
NAD+/NADH imbalance in DKD manifests as NAD+ decline and NADH overload, which aggravates oxidative stress mediated by ROC in the kidneys and causes mitochondrial dysfunction, energy metabolic disorders and inactivation of various NAD+-dependent enzymes [
51]. In a study, impaired NAD+ synthesis in mice with CKD was associated with decreased expression of key enzymes, including quinolinate phosphoribosyl transferase (QPRT) in the NAD+ de novo synthesis pathway and nicotinamide nucleotide adenylyl transferase 1 (NMNAT1) and NMNAT 3 in the salvage synthesis pathway [
54]. In this study, the expression of NAMN (precursor to the NAD+ de novo synthesis pathway) and niacinamide (NAM, precursor to the salvage synthesis pathway) was increased after EMPA treatment, indicating that these key enzymes may act as targets for EMPA to amend energy metabolism. In addition, most NAM is mainly produced by NAD+-consuming enzymes, including the superfamily members of sirtuins (SIRT), poly (ADP-ribose) polymerase (PARP) and CD38 [
55]. However, in a study, canagliflozin treatment reversed the reduction in SIRT1 expression in db/db mice and improved local NAD+ metabolism in the kidney [
56]. We believe this study will provide novel insights into the renoprotection mechanisms of SGLT2 inhibitors.
As shown in Fig.
7, we discovered a dominant accumulation of metabolites after purine degradation in db/db mice, such as xanthine, hypoxanthine and uric acid. However, metabolites involved in ATP degradation-mediated purine nucleotide conversion pathway, including adenosine diphosphate (ADP), adenosine monophosphate (AMP), adenosine (ADO) and adenine (AD), were downregulated after EMPA treatment. EMPA blocks the catabolism of hypoxanthine but improves the recycling of hypoxanthine, which is consistent with the amelioration of ischemia–reperfusion renal injury in mice after intervention with XOR inhibitors [
57]. Previous studies have confirmed that the uric acid-decreasing effect of SGLT2 inhibitors is associated with its promoting effects on uric acid excretion and reabsorption inhibition [
58,
59]. Based on the data of this study, we speculate that EMPA may reduce uric acid by blocking purine degradation (similar to XOR inhibitors).
Inhibition of the uric acid generation pathway promotes the increase of ATP degradation-mediated purine nucleotide conversion pathway. In the kidneys, the local paracrine release of purine nucleotides is regulated by connexins [
60]. Abnormal expression of connexins and the subsequent interruption of intercellular communication in DKD result in the up-regulated expression and activity of hemichannels, which mediate the imbalances for the release of ATP and ADO and may have contributed in part to the pathology of DKD [
61‐
63]. ATP and its degradation metabolite ADO are effective extracellular signaling molecules for purinergic signaling in the kidney, which activate P2 and P1 receptors, respectively, to perform contradictory physiological functions, such as the pro-inflammatory response for ATP and the anti-inflammatory response for ADO [
32]. Downregulation of ADO and its receptor A1R is involved in ultrafiltration in the early stages of DKD [
64]. A2AR (an adenosine receptor) agonists can attenuate proteinuria and reduce the number of pro-inflammatory cytokines in DKD [
65]. Moreover, ADO can be produced by NAD+ metabolism, and enhancing NAD+ metabolism-mediated ADO production has been shown to prevent ischemia-induced acute kidney injury [
66]. Researchers have demonstrated that blocking CX43 and its mediated local ATP release from renal tubular epithelial cells can ameliorate renal fibrosis [
67]. Interestingly, a recent study observed that EMPA treatment repaired ventricular myocytes' gap junctional intercellular communication and attenuated ventricular fibrosis in mice with metabolic syndrome [
68]. Unfortunately, no studies have explored the effects of SGLT2 inhibitors on renal CX43. However, this is undoubtedly an interesting research direction, as the modulation of the local purinergic system by SGLT2 inhibitors may be optimized by using nanotechnology to embed drugs with Cx43 hemichannel blocking effects into nanomaterials with specific targeting to the renal tubules.
A previous study described kidney-specific metabolic reprogramming associated with mitochondrial dysfunctional in db/db mice, manifesting as a compensatory increase in glycolysis and fatty acid metabolism, which was a response to diminished production of ATP induced by dysfunction of the mitochondrial electron transport chain and uncoupling of oxidative phosphorylation [
69]. Dihydroorotate dehydrogenase (DHODH) is the first rate-limiting enzyme of pyrimidine de novo synthesis, catalyzing the dihydroorotate oxidized to orotate and further producing various downstream pyrimidine nucleotides [
70]. DHODH delivers electrons to ubiquinone during this process and provides reductive ubiquinone for compounds I and III of the respiratory chain, thus coupling the pyrimidine metabolism with mitochondrial phosphorylation [
71]. It has been demonstrated that the knockdown of intracellular DHODH partially inhibits the activity of respiratory chain complex III and increases mitochondrial ROS production [
72]. However, DHODH inhibitors decline the level of pyrimidine nucleotide but increase the level of upstream metabolites of dihydroorotate [
73]. The results of the abovementioned studies are consistent with those observed in this study (the kidney of db/db mice). Furthermore, we found that dihydroorotate levels were significantly increased, whereas the levels of its downstream metabolites were increased, including orotate (despite P = 0.052, it had an increasing trend, with VIP = 1.28 and FC = 1.45), uridine monophosphate (UMP), uracil dinucleotide (UDP) and cytidine monophosphate (CMP). However, the metabolites uridine triphosphate (UTP) and cytidine triphosphate (CTP) involved in the pyrimidine salvage pathway were not detected. Therefore, we speculate that EMPA improves mitochondrial dysfunction and alleviates the metabolic reprogramming of the kidneys in DKD by promoting DHODH-mediated de novo synthesis of pyrimidine to increase mitochondrial electron transport.
Furthermore, in this study, metabolic alterations were observed less in the serum than in the kidney after EMPA treatment. The potential interfered pathway of serum metabolites was ‘glycine, serine and threonine metabolism’, specifically involving the biological transformation of
d-serine and
l-serine.
d-Serine, the most abundant
d-amino acid in mammals, is produced from
l-serine mediated by serine racemase. The kidney mostly regulates
d-serine levels by excreting it through urine or degrading it via
d-amino acid oxidase (DAAO, highly expressed in the kidney). However, the intermediate metabolites during
d-serine oxidative decomposition, mainly H
2O
2, have strong renal toxicity. Previous studies have demonstrated that
d-serine concentration is positively correlated with GFR and can serve as a clinical diagnostic biomarker for CKD [
74]. However, Tomonori Kimura et al. discovered that the level of
d-serine did not increase with a decline in GFR in a proportion of patients with CKD [
75]. In this study, we found that
d-serine levels increased in serum but were reduced in the kidney in db/db mice. Moreover, the ratio of
l-serine to
d-serine did not change before and after EMPA treatment (Fig.
7), which is consistent with the results of the aforementioned study [
75], collectively indicating that a compensation mechanism in the kidney maintains a certain ratio of
l-serine to
d-serine. We also found that EMPA significantly reduced the level of local
d-serine in the kidney, indicating that it is associated with improving renal function or alterations in
l-serine metabolism.
Our study has some limitations. First, the untargeted metabolomics we used is a relative quantification method of the metabolites and needs to be followed up with target validation. Second, the sample size of our study is small, and more animal or clinical samples can be included in the follow-up and combined with some new histological techniques (e.g., spatial omics techniques) for the in-depth study.
In conclusion, this study demonstrated conspicuous metabolic reprogramming in mice with DKD. EMPA treatment improved kidney function and morphology by regulating metabolic reprogramming, including regulation of renal reductive stress, alleviation of mitochondrial dysfunction and reduction in renal oxidative stress reaction. Therefore, this study provides an essential reference for understanding the mechanism of EMPA in renoprotection.