Obesity is considered a significant risk factor for the progression of chronic kidney disease to end-stage renal disease [4]. Studies suggest that obesity-related kidney injury can occur in the early stages of obesity, where lipids may lead to damage in glomerular and tubular cells, subsequently promoting the progression of kidney disease [16]. Therefore, early prevention and treatment of obesity-related kidney injury are of crucial importance in slowing the progression of chronic kidney disease. In this study, we employed a network pharmacology approach to explore the potential molecular mechanisms of diosmin in obesity-related kidney injury. Through PPI network and molecular docking analyses, we identified 10 key targets—AKT1, TNF-α, SRC, EGFR, ESR1, CASP3, MMP9, PPARG, GSK3B, and MMP2—as crucial in the treatment of obesity-related kidney injury. Furthermore, validation of these 10 core genes was conducted in a palmitic acid induced HK2 cell injury model. We observed that these core genes are primarily involved in the biological processes of cell apoptosis, inflammation, and oxidative stress.
The GSK3B gene encodes glycogen synthase kinase 3 beta, which also regulates glycogen metabolism and cell apoptosis [17]. GSK3B is considered a key regulatory factor in cell apoptosis [18]. Numerous studies have shown that GSK3B plays a crucial role in the pathogenesis of acute kidney injury, and chemical inhibition or genetic knockout of GSKB has a renal protective effect in acute kidney injury [19]. In mice exposed to high doses of diclofenac, a selective inhibitor of GSK3B called TDZD-8 can prevent tubular necrosis and apoptosis, improving overall survival and kidney function [20]. CASP3 encodes cysteine-aspartate lyase and plays a central role in the initiation and execution stages of cell apoptosis [21]. CASP3 is also an important upstream regulatory factor in ischemia-reperfusion kidney injury and kidney fibrosis [22]. AKT1 is a serine/threonine kinase, an important mediator in various signaling cascades, activated in response to phosphatidylinositol 3-kinase [23]. It can mediate various biological functions, including metabolism, proliferation, and apoptosis [23]. AKT1 has been reported to be involved in various processes of acute kidney injury and chronic kidney disease [24]. Previous research indicates that in the transition from acute kidney injury to chronic kidney disease, knockout of AKT1 inhibits the activation of GSK3B and the activation of CASP3 in the acute phase, thereby reducing tubular apoptosis [25]. However, in the chronic phase, it exhibits the opposite effect, promoting tubular apoptosis [25]. Overall, AKT1, CASP3, and GSK3B interact in cell apoptosis, forming a complex regulatory network. AKT1 exerts anti-apoptotic or pro-apoptotic effects by inhibiting or promoting the activity of CASP3 and GSK3B. Meanwhile, GSK3B also participates in regulating the process of cell apoptosis. In our study, we found that diosmin inhibits the gene expression levels of GSK3B, CASP3, and AKT1 in palmitic acid induced HK2 cells. This suggests that diosmin may play a protective role in obesity-related kidney injury by interacting with GSK3B, CASP3, and AKT1 to regulate cell apoptosis. Furthermore, molecular docking indicates that diosmin has optimal binding affinity with GSK3B, suggesting that GSK3B may be a more crucial target for treating obesity-related kidney injury.
PPARG is a nuclear hormone receptor that controls the transcription of multiple specific genes, participating in the regulation of various biological processes, including lipid metabolism, insulin resistance, inflammation, oxidative stress, and cell apoptosis, playing a crucial role in renal physiology and diseases [26]. PPARG plays a significant role in obesity-related kidney injury [27]. A study confirmed that in leptin deficient obese mice, the knockout of PPARG triggered insulin resistance and inflammatory responses, exacerbating kidney damage [28]. Another study indicated that palmitic acid induced downregulation of PPARG expression in podocytes resulted in renal inflammation and oxidative stress injury, affecting glomerular filtration function [29]. Abundant research has demonstrated that PPARG agonists protect the kidneys by enhancing insulin sensitivity and inhibiting inflammation [27]. EGFR is a member of the receptor tyrosine kinase family expressed in various cell types in the adult mammalian kidney, including podocytes, endothelial cells, and mesangial cells [30]. Studies have shown that persistent activation of tubular EGFR promotes infiltration of inflammatory cells and fibroblast proliferation and migration, leading to the progression of renal fibrosis [30,31]. ESR1 encodes the estrogen receptor, which can directly bind to the promoter sequence of the NLRP3 inflammasome, regulating NLRP3 transcription, activating Caspase1, and inducing inflammatory responses [32]. SRC, as an oncogene regulating various biological functions, including mitochondrial function, plays a crucial role in acute and chronic kidney injury [33]. As a protein tyrosine kinase, SRC can regulate NF-κB p65 and MAPKs, serving as an important molecule in the renal inflammatory cascade response to lipopolysaccharide induced acute kidney injury [34]. Another study suggested that the SRC family kinase inhibitor PP2 could improve the inflammatory response and oxidative stress induced by lipopolysaccharide in acute kidney injury [33]. Matrix metalloproteinases (MMPs) are enzymes belonging to the zinc metalloproteinase family subfamily, responsible for the degradation of extracellular matrix [35]. Ischemic kidney injury can induce upregulation of MMP2 and MMP9, which, by disrupting the perivascular matrix, worsen inflammation and contribute to kidney damage [36]. TNF-α is a cytokine with pro-inflammatory and immune-regulatory effects, closely associated with the occurrence and development of kidney injury [37]. Synthesized by intrinsic glomerular cells and tubular cells, TNF-α can induce cells to release pro-inflammatory chemokines in an autocrine and paracrine manner, amplifying renal inflammatory responses [37]. In a rat model of immune-mediated glomerulonephritis induced by anti-glomerular basement membrane antibodies, systemic administration of TNF-α increased neutrophil infiltration, glomerular capillary thrombosis, and the incidence of proteinuria, thereby worsening the severity of glomerular injury [38]. TNF-α blockade reduced proteinuria, inflammation, and kidney damage in the rat glomerulonephritis model [39]. In summary, PPARG, EGFR, ESR1, SRC, MMP2, MMP9, and TNF-α play diverse roles in inflammation and oxidative stress, regulating the occurrence and development of inflammatory responses through complex interaction networks.
The results of this study suggest that diosmin inhibits the gene expression levels of EGFR, ESR1, MMP2, MMP9, and TNF-α in palmitic acid induced HK2 cells while promoting PPARG gene expression. This indicates its potential intervention in obesity-related kidney injury by modulating inflammatory responses and oxidative stress. Unfortunately, no changes in SRC gene expression were observed, inconsistent with the predictions of network pharmacology. This discrepancy may be attributed to: (a) Network pharmacology relies on computational models and structural information, unable to fully reflect the complex physiological environment within the organism. (b) The complex intracellular environment may lead to changes in the interaction between drugs and target receptors. SRC, as a protein tyrosine kinase, may exhibit different regulatory effects in different cell lines or cellular environments.
According to KEGG terminology, the therapeutic targets of diosmin against obesity-related kidney injury are primarily associated with the AGE-RAGE, PI3K-AKT, and PPAR signaling pathways. The AGE-RAGE signaling pathway plays a crucial role in various acute and chronic kidney injuries [41]. Interaction between AGE and RAGE activates the nuclear factor NF-κB, leading to cellular dysfunction and increased production and release of inflammatory cytokines [42]. Research also indicates that the AGE-RAGE complex can specifically activate the p21 protein, stimulating signaling pathways such as JAK1&2/STAT1, MAPK, and NADPH, which directly and indirectly generate reactive oxygen species, causing inflammation and endothelial dysfunction, thereby exacerbating kidney damage [43]. The PI3K-AKT signaling pathway is an essential intracellular signaling cascade that plays a significant role in obesity-related kidney injury by regulating various proteins [44]. Research indicates that arbutin mitigates lipopolysaccharide-induced acute kidney injury by suppressing inflammation and cell apoptosis through the PI3K/AKT/NRF2 pathway [45]. It has been reported that in fatty acid and alcohol-induced podocyte and HK2 cell injury, modulation of the PPAR pathway can activate the NF-κB/NLRP3 mediated immune and inflammatory response, resulting in kidney injury [30,46]. Therefore, the AGE-RAGE, PI3K-AKT, and PPAR signaling pathways are closely associated with the occurrence and development of obesity-related kidney injury.
In conclusion, this research presents an optimized approach to elucidate the pharmacological mechanisms of diosmin and introduces a novel candidate for treating obesity-related kidney injury. However, the study has certain limitations. Firstly, key target gene expression levels were only validated in vitro, potentially leading to slightly skewed results. Secondly, the connections between drug-target-related signaling pathways were not verified. In the future, we aim to refine experimental designs and validate our findings through both in vivo and in vitro experiments.