Integrated Network Pharmacology Analysis and Experimental Validation to Elucidate the Mechanism of Acteoside in Treating Diabetic Kidney Disease.

Background: Acteoside, an active ingredient found in various medicinal herbs, is effective in the treatment of diabetic kidney disease (DKD); however, the intrinsic pharmacological mechanism of action of acteoside in the treatment of DKD remains unclear. This study utilizes a combined approach of network pharmacology and experimental validation to investigate the potential molecular mechanism systematically. Methods: First, acteoside potential targets and DKD-associated targets were aggregated from public databases. Subsequently, utilizing protein-protein interaction (PPI) networks, alongside GO and KEGG pathway enrichment analyses, we established target-pathway networks to identify core potential therapeutic targets and pathways. Further, molecular docking facilitated the confirmation of interactions between acteoside and central targets. Finally, the conjectured molecular mechanisms of acteoside against DKD were verified through experimentation on unilateral nephrectomy combined with streptozotocin (STZ) rat model. The underlying downstream mechanisms were further investigated. Results: Network pharmacology identified 129 potential intersected targets of acteoside for DKD treatment, including targets such as AKT1, TNF, Casp3, MMP9, SRC, IGF1, EGFR, HRAS, CASP8, and MAPK8. Enrichment analyses indicated the PI3K-Akt, MAPK, Metabolic, and Relaxin signaling pathways could be involved in this therapeutic context. Molecular docking revealed high-affinity binding of acteoside to PIK3R1, AKT1, and NF-κB1. In vivo studies validated the therapeutic efficacy of acteoside, demonstrating reduced blood glucose levels, improved serum Scr and BUN levels, decreased 24-hour urinary total protein (P<0.05), alongside mitigated podocyte injury (P<0.05) and ameliorated renal pathological lesions. Furthermore, this finding indicates that acteoside inhibits the expression of pyroptosis markers NLRP3, Caspase-1, IL-1ß, and IL-18 through the modulation of the PI3K/AKT/NF-κB pathway. Conclusion: Acteoside demonstrates renoprotective effects in DKD by regulating the PI3K/AKT/NF-κB signaling pathway and alleviating pyroptosis. This study explores the pharmacological mechanism underlying acteoside's efficacy in DKD treatment, providing a foundation for further basic and clinical research.

c-Cbl induced podocin ubiquitination contributes to the podocytes injury in diabetic nephropathy.

The ubiquitination function in diabetic nephropathy (DN) has attracted much attention, but there is a lack of information on its ubiquitylome profile. To examine the differences in protein content and ubiquitination in the kidney between db/db mice and db/m mice, we deployed liquid chromatography-mass spectrometry (LC-MS/MS) to conduct analysis. We determined 145 sites in 86 upregulated modified proteins and 66 sites in 49 downregulated modified proteins at the ubiquitinated level. Moreover, 347 sites among the 319 modified proteins were present only in the db/db mouse kidneys, while 213 sites among the 199 modified proteins were present only in the db/m mouse kidneys. The subcellular localization study indicated that the cytoplasm had the highest proportion of ubiquitinated proteins (31.87%), followed by the nucleus (30.24%) and the plasma membrane (20.33%). The enrichment analysis revealed that the ubiquitinated proteins are mostly linked to tight junctions, oxidative phosphorylation, and thermogenesis. Podocin, as a typical protein of slit diaphragm, whose loss is a crucial cause of proteinuria in DN. Consistent with the results of ubiquitination omics, the K261R mutant of podocin induced the weakest ubiquitination compared with the K301R and K370R mutants. As an E3 ligase, c-Cbl binds to podocin, and the regulation of c-Cbl can impact the ubiquitination of podocin. In conclusion, in DN, podocin ubiquitination contributes to podocyte injury, and K261R is the most significant site. c-Cbl participates in podocin ubiquitination and may be a direct target for preserving the integrity of the slit diaphragm structure, hence reducing proteinuria in DN.

miR-4645-3p attenuates podocyte injury and mitochondrial dysfunction in diabetic kidney disease by targeting Cdk5.

Podocyte injury plays a critical role in the progression of diabetic kidney disease (DKD), but the underlying cellular and molecular mechanisms remain poorly understanding. MicroRNAs (miRNAs) can disrupt gene expression by inducing translation inhibition and mRNA degradation, and recent evidence has shown that miRNAs may play a key role in many kidney diseases. In this study, we identified miR-4645-3p by global transcriptome expression profiling as one of the major downregulated miRNAs in high glucose-cultured podocytes. Moreover, whether DKD patients or STZ-induced diabetic mice, expression of miR-4645-3p was also significantly decreased in kidney. In the podocytes cultured by normal glucose, inhibition of miR-4645-3p expression promoted mitochondrial damage and podocyte apoptosis. In the podocytes cultured by high glucose (30 mM glucose), overexpression of miR-4645-3p significantly attenuated mitochondrial dysfunction and podocyte apoptosis induced by high glucose. Furthermore, we found that miR-4645-3p exerted protective roles by targeting Cdk5 inhibition. In vitro, miR-4645-3p obviously antagonized podocyte injury by inhibiting overexpression of Cdk5. In vivo of diabetic mice, podocyte injury, proteinuria, and impaired renal function were all effectively ameliorated by treatment with exogenous miR-4645-3p. Collectively, these findings demonstrate that miR-4645-3p can attenuate podocyte injury and mitochondrial dysfunction in DKD by targeting Cdk5. Sustaining the expression of miR-4645-3p in podocytes may be a novel strategy to treat DKD.

Application and design considerations of ROS-based nanomaterials in diabetic kidney disease.

Diabetic nephropathy (DKD) is a common chronic complication of diabetes mellitus and an important cause of cardiovascular-related death. Oxidative stress is a key mechanism leading to diabetic nephropathy. However, the current main therapeutic approach remains combination therapy and lacks specific therapies targeting oxidative stress. With the development of nanotechnology targeting ROS, therapeutic fluids regarding their treatment of diabetic nephropathy have attracted attention. In this review, we provide a brief overview of various ROS-based nanomaterials for DKD, including ROS-scavenging nanomaterials, ROS-associated nanodelivery materials, and ROS-responsive nanomaterials. In addition, we summarize and discuss key factors that should be considered when designing ROS-based nanomaterials, such as biosafety, efficacy, targeting, and detection and monitoring of ROS.

Lipid metabolism disorder in diabetic kidney disease.

Diabetic kidney disease (DKD), a significant complication associated with diabetes mellitus, presents limited treatment options. The progression of DKD is marked by substantial lipid disturbances, including alterations in triglycerides, cholesterol, sphingolipids, phospholipids, lipid droplets, and bile acids (BAs). Altered lipid metabolism serves as a crucial pathogenic mechanism in DKD, potentially intertwined with cellular ferroptosis, lipophagy, lipid metabolism reprogramming, and immune modulation of gut microbiota (thus impacting the liver-kidney axis). The elucidation of these mechanisms opens new potential therapeutic pathways for DKD management. This research explores the link between lipid metabolism disruptions and DKD onset.

[Exercise promotes irisin expression to ameliorate renal injury in type 2 diabetic rats].

OBJECTIVE: To investigate the role of irisin in exercise-induced improvement of renal function in type 2 diabetic rats. METHODS: Forty male SD rats aged 4-6 weeks were randomized into normal control group, type 2 diabetes mellitus model group, diabetic exercise (DE) group and diabetic irisin (DI) group (n=8). The rats in DE group were trained with treadmill running for 8 weeks, and those in DI group were given scheduled irisin injections for 8 weeks. After the treatments, blood biochemical parameters of the rats were examined, and renal histopathology was observed with HE, Masson and PAS staining. Western blotting was used to detect the protein expression levels in the rats'kidneys. RESULTS: The diabetic rats showed significantly increased levels of fasting insulin, total cholesterol, triglyceride, serum creatinine and blood urea nitrogen with lowered serum irisin level (all P < 0.05). Compared with those in DM group, total cholesterol, triglyceride, serum creatinine and blood urea nitrogen levels were decreased and serum irisin levels were increased in both DE and DI groups (all P < 0.05). The rats in DM group showed obvious structural disorders and collagen fiber deposition in the kidneys, which were significantly improved in DE group and DI group. Both regular exercises and irisin injections significantly ameliorated the reduction of FNDC5, LC3-II/I, Atg7, Beclin-1, p-AMPK, AMPK and SIRT1 protein expressions and lowered of p62 protein expression in the kidneys of the diabetic rats (all P < 0.05). CONCLUSION: Both exercise and exogenous irisin treatment improve nephropathy in type 2 diabetic rats possibly due to irisin-mediated activation of the AMPK/SIRT1 pathway in the kidneys to promote renal autophagy.

Scopoletin alleviates high glucose-induced toxicity in human renal proximal tubular cells via inhibition of oxidative damage, epithelial-mesenchymal transition, and fibrogenesis.

BACKGROUND: Recent years of evidence suggest the crucial role of renal tubular cells in developing diabetic kidney disease. Scopoletin (SCOP) is a plant-based coumarin with numerous biological activities. This study aimed to determine the effect of SCOP on renal tubular cells in developing diabetic kidney disease and to elucidate mechanisms. METHODS AND RESULTS: In this study, SCOP was evaluated in vitro using renal proximal tubular (HK-2) cells under hyperglycemic conditions to understand its mechanism of action. In HK-2 cells, SCOP alleviated the high glucose-generated reactive oxygen species (ROS), restored the levels of reduced glutathione, and decreased lipid peroxidation. High glucose-induced alteration in the mitochondrial membrane potential was markedly restored in the SCOP-treated cells. Moreover, SCOP significantly reduced the high glucose-induced apoptotic cell population in the Annexin V-FITC flow cytometry study. Furthermore, high glucose markedly elevated the mRNA expression of fibrotic and extracellular matrix (ECM) components, namely, transforming growth factor (TGF)-ß, alfa-smooth muscle actin (α-SMA), collagen I, and collagen III, in HK-2 cells compared to the untreated cells. SCOP treatment reduced these mRNA expressions compared to the high glucose-treated cells. Collagen I and TGF-ß protein levels were also significantly reduced in the SCOP-treated cells. Further findings in HK-2 cells revealed that SCOP interfered with the epithelial-mesenchymal transition (EMT) in the high glucose-treated HK-2 cells by normalizing E-cadherin and downregulating the vimentin and α-SMA proteins. CONCLUSIONS: In conclusion, SCOP modulates the high glucose-generated renal tubular cell oxidative damage and accumulation of ECM components and may be a promising molecule against diabetic nephropathy.

Research progress on the role of extracellular vesicles in the pathogenesis of diabetic kidney disease.

Diabetic kidney disease (DKD) is a serious complication of diabetes mellitus (DM) and has become the main cause of end-stage renal disease worldwide. In recent years, with the increasing incidence of DM, the pathogenesis of DKD has received increasing attention. The pathogenesis of DKD is diverse and complex. Extracellular vesicles (EVs) contain cell-derived membrane proteins, nucleic acids (such as DNA and RNA) and other important cellular components and are involved in intercellular information and substance transmission. In recent years, an increasing number of studies have confirmed that EVs play an important role in the development of DKD. The purpose of this paper is to explain the potential diagnostic value of EVs in DKD, analyze the mechanism by which EVs participate in intercellular communication, and explore whether EVs may become drug carriers for targeted therapy to provide a reference for promoting the implementation and application of exosome therapy strategies in clinical practice.

Identification of Lipotoxicity-Related Biomarkers in Diabetic Nephropathy Based on Bioinformatic Analysis.

Objective: This study is aimed at investigating diagnostic biomarkers associated with lipotoxicity and the molecular mechanisms underlying diabetic nephropathy (DN). Methods: The GSE96804 dataset from the Gene Expression Omnibus (GEO) database was utilized to identify differentially expressed genes (DEGs) in DN patients. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted using the DEGs. A protein-protein interaction (PPI) network was established to identify key genes linked to lipotoxicity in DN. Immune infiltration analysis was employed to identify immune cells with differential expression in DN and to assess the correlation between these immune cells and lipotoxicity-related hub genes. The findings were validated using the external dataset GSE104954. ROC analysis was performed to assess the diagnostic performance of the hub genes. The Gene set enrichment analysis (GSEA) enrichment method was utilized to analyze the key genes associated with lipotoxicity as mentioned above. Result: In this study, a total of 544 DEGs were identified. Among them, extracellular matrix (ECM), fatty acid metabolism, AGE-RAGE, and PI3K-Akt signaling pathways were significantly enriched. Combining the PPI network and lipotoxicity-related genes (LRGS), LUM and ALB were identified as lipotoxicity-related diagnostic biomarkers for DN. ROC analysis showed that the AUC values for LUM and ALB were 0.882 and 0.885, respectively. The AUC values for LUM and ALB validated in external datasets were 0.98 and 0.82, respectively. Immune infiltration analysis revealed significant changes in various immune cells during disease progression. Macrophages M2, mast cells activated, and neutrophils were significantly associated with all lipotoxicity-related hub genes. These key genes were enriched in fatty acid metabolism and extracellular matrix-related pathways. Conclusion: The identified lipotoxicity-related hub genes provide a deeper understanding of the development mechanisms of DN, potentially offering new theoretical foundations for the development of diagnostic biomarkers and therapeutic targets related to lipotoxicity in DN.

Not too much, not too little-just the right amount: the story of YAP in the podocyte.

Chen et al. identify dysregulation of the transcriptional activator Yes-associated protein in the podocytes of diabetic mouse and human kidneys. Podocyte Yes-associated protein deficiency led to downregulation of the key transcription factor Wilms' tumor 1, and worsened podocyte injury in a mouse model of diabetic kidney injury. Yes-associated protein may therefore play a critical role in diabetic podocyte injury via regulation of Wilms' tumor 1 expression.

Unraveling the role of natriuretic peptide clearance receptor (NPR3) in glomerular diseases.

Natriuretic peptides (NPs) are cardio-derived hormones that have a crucial role in maintaining cardiovascular homeostasis. Physiological effects of NPs are mediated by binding to natriuretic peptide receptors 1 and 2 (NPR1/2), whereas natriuretic peptide receptor 3 (NPR3) acts as a clearance receptor that removes NPs from the circulation. Mouse studies have shown that local NP-signaling in the kidney glomerulus is important for the maintenance of renal homeostasis. In this study we examined the expression of NPR3 in kidney tissue and explored its involvement in renal physiology and disease by generating podocyte-specific knockout mice (NPR3podKO) as well as by using an NPR3 inhibitor (NPR3i) in rodent models of kidney disease. NPR3 was highly expressed by podocytes. NPR3podKO animals showed no renal abnormalities under healthy conditions and responded similarly to nephrotoxic serum (NTS) induced glomerular injury. However, NPR3i showed reno-protective effects in the NTS-induced model evidenced by decreased glomerulosclerosis and reduced podocyte loss. In a ZSF1 rat model of diabetic kidney injury, therapy alone with NPR3i did not have beneficial effects on renal function/histology, but when combined with losartan (angiotensin receptor blocker), NPR3i potentiated its ameliorative effects on albuminuria. In conclusion, these results suggest that NPR3 may contribute to kidney disease progression.

Pharmacological Blockade of the Adenosine A2B Receptor Is Protective of Proteinuria in Diabetic Rats, through Affecting Focal Adhesion Kinase Activation and the Adhesion Dynamics of Podocytes.

Induction of the adenosine receptor A2B (A2BAR) expression in diabetic glomeruli correlates with an increased abundance of its endogenous ligand adenosine and the progression of kidney dysfunction. Remarkably, A2BAR antagonism protects from proteinuria in experimental diabetic nephropathy. We found that A2BAR antagonism preserves the arrangement of podocytes on the glomerular filtration barrier, reduces diabetes-induced focal adhesion kinase (FAK) activation, and attenuates podocyte foot processes effacement. In spreading assays using human podocytes in vitro, adenosine enhanced the rate of cell body expansion on laminin-coated glass and promoted peripheral pY397-FAK subcellular distribution, while selective A2BAR antagonism impeded these effects and attenuated the migratory capability of podocytes. Increased phosphorylation of the Myosin2A light chain accompanied the effects of adenosine. Furthermore, when the A2BAR was stimulated, the cells expanded more broadly and more staining of pS19 myosin was detected which co-localized with actin cables, suggesting increased contractility potential in cells planted onto a matrix with a stiffness similar to of the glomerular basement membrane. We conclude that A2BAR is involved in adhesion dynamics and contractile actin bundle formation, leading to podocyte foot processes effacement. The antagonism of this receptor may be an alternative to the intervention of glomerular barrier deterioration and proteinuria in the diabetic kidney disease.

Deletion of IRE1α in podocytes exacerbates diabetic nephropathy in mice.

Protein misfolding in the endoplasmic reticulum (ER) of podocytes contributes to the pathogenesis of glomerular diseases. Protein misfolding activates the unfolded protein response (UPR), a compensatory signaling network. We address the role of the UPR and the UPR transducer, inositol-requiring enzyme 1α (IRE1α), in streptozotocin-induced diabetic nephropathy in mice. Diabetes caused progressive albuminuria in control mice that was exacerbated in podocyte-specific IRE1α knockout (KO) mice. Compared to diabetic controls, diabetic IRE1α KO mice showed reductions in podocyte number and synaptopodin. Glomerular ultrastructure was altered only in diabetic IRE1α KO mice; the major changes included widening of podocyte foot processes and glomerular basement membrane. Activation of the UPR and autophagy was evident in diabetic control, but not diabetic IRE1α KO mice. Analysis of human glomerular gene expression in the JuCKD-Glom database demonstrated induction of genes associated with the ER, UPR and autophagy in diabetic nephropathy. Thus, mice with podocyte-specific deletion of IRE1α demonstrate more severe diabetic nephropathy and attenuation of the glomerular UPR and autophagy, implying a protective effect of IRE1α. These results are consistent with data in human diabetic nephropathy and highlight the potential for therapeutically targeting these pathways.

Leonuride protects diabetic nephropathy by activating SREBPs pathway.

Diabetic nephropathy (DN), a micro vascular complication of diabetes, is the main cause of end-stage renal disease, with a morbidity over 40% of diabetes. High glucose and lipid metabolism dysfunction are the leading cause of the development of DN. Previous study demonstrated that increased expression or activation of SREBPs in models of DN. Leonuride (LE), as an active constituent of Leonurus japonicus Houttuyn, has multiple biological activities, including antioxidant and anti-inflammatory effects. Previous studies showed that increasing the degradation of mature SREBPs is a robust way of lowering lipids and improve lipid metabolism dysfunction. However, effective regulation method of SREBPs degradation are still lacking. Herein, this study indicated that LE can effectively improve glucose and lipid metabolism disorders. In addition, the kidney function was also improved by inhibition of SREBPs activities in streptozocin (STZ)-induced type II diabetic mice. To our knowledge, this is the first time to describe the detailed mechanism of LE on the inhibition of precursor SREBPs, which would present a new direction for diabetic nephropathy treatment.

Unveiling the crucial role of intercellular adhesion molecule-1 in secondary diabetic complications.

Diabetes mellitus is associated with secondary complications such as diabetic retinopathy (DR), nephropathy (DN), and cardiomyopathy (DCM), all of which significantly impact patient health. Intercellular adhesion molecule-1 (ICAM-1) has been implicated in inflammatory responses and endothelial dysfunction, both crucial in the pathogenesis of these complications. The goal of this review is to investigate at potential therapy methods that target ICAM-1 pathways and to better understand the multifaceted role of ICAM-1 in secondary diabetic problems. A meticulous analysis of scholarly literature published globally was conducted to examine ICAM-1involvement in inflammatory processes, endothelial dysfunction, and oxidative stress related to diabetes and its complications. Elevated ICAM-1 levels are strongly associated with augmented leukocyte adhesion, compromised microvascular function, and heightened oxidative stress in diabetes. These pathways contribute significantly to DR, DN, and DCM pathogenesis, highlighting ICAM-1 as a key player in their progression. Understanding ICAM-1 role in secondary diabetic complications offers insights into novel therapeutic strategies. Targeting ICAM-1 pathways may mitigate inflammation, improve endothelial function, and ultimately attenuate diabetic complications, thereby enhancing patient health outcomes. Continued research in this area is crucial for developing effective targeted therapies.

Isocitrate dehydrogenase 1 upregulation in urinary extracellular vesicles from proximal tubules of type 2 diabetic rats.

Diabetic nephropathy (DN) is a major cause of chronic kidney disease. Microalbuminuria is currently the most common non-invasive biomarker for the early diagnosis of DN. However, renal structural damage may have advanced when albuminuria is detected. In this study, we sought biomarkers for early DN diagnosis through proteomic analysis of urinary extracellular vesicles (uEVs) from type 2 diabetic model rats and normal controls. Isocitrate dehydrogenase 1 (IDH1) was significantly increased in uEVs from diabetic model rats at the early stage despite minimal differences in albuminuria between the groups. Calorie restriction significantly suppressed the increase in IDH1 in uEVs and 24-hour urinary albumin excretion, suggesting that the increase in IDH1 in uEVs was associated with the progression of DN. Additionally, we investigated the origin of IDH1-containing uEVs based on their surface sugar chains. Lectin affinity enrichment and immunohistochemical staining showed that IDH1-containing uEVs were derived from proximal tubules. These findings suggest that the increase in IDH1 in uEVs reflects pathophysiological alterations in the proximal tubules and that IDH1 in uEVs may serve as a potential biomarker of DN in the proximal tubules.

Gut microbiota microbial metabolites in diabetic nephropathy patients: far to go.

Diabetic nephropathy (DN) is one of the main complications of diabetes and a major cause of end-stage renal disease, which has a severe impact on the quality of life of patients. Strict control of blood sugar and blood pressure, including the use of renin-angiotensin-aldosterone system inhibitors, can delay the progression of diabetic nephropathy but cannot prevent it from eventually developing into end-stage renal disease. In recent years, many studies have shown a close relationship between gut microbiota imbalance and the occurrence and development of DN. This review discusses the latest research findings on the correlation between gut microbiota and microbial metabolites in DN, including the manifestations of the gut microbiota and microbial metabolites in DN patients, the application of the gut microbiota and microbial metabolites in the diagnosis of DN, their role in disease progression, and so on, to elucidate the role of the gut microbiota and microbial metabolites in the occurrence and prevention of DN and provide a theoretical basis and methods for clinical diagnosis and treatment.

Protective effect of phospholipids in lipoproteins against diabetic kidney disease: A Mendelian randomization analysis.

BACKGROUND: The etiology of diabetic kidney disease is complex, and the role of lipoproteins and their lipid components in the development of the disease cannot be ignored. However, phospholipids are an essential component, and no Mendelian randomization studies have yet been conducted to examine potential causal associations between phospholipids and diabetic kidney disease. METHODS: Relevant exposure and outcome datasets were obtained through the GWAS public database. The exposure datasets included various phospholipids, including those in LDL, IDL, VLDL, and HDL. IVW methods were the primary analytical approach. The accuracy of the results was validated by conducting heterogeneity, MR pleiotropy, and F-statistic tests. MR-PRESSO analysis was utilized to identify and exclude outliers. RESULTS: Phospholipids in intermediate-density lipoprotein (OR: 0.8439; 95% CI: 0.7268-0.9798), phospholipids in large low- density lipoprotein (OR: 0.7913; 95% CI: 0.6703-0.9341), phospholipids in low- density lipoprotein (after removing outliers, OR: 0.788; 95% CI: 0.6698-0.9271), phospholipids in medium low- density lipoprotein (OR: 0.7682; 95% CI: 0.634-0.931), and phospholipids in small low-density lipoprotein (after removing outliers, OR: 0.8044; 95% CI: 0.6952-0.9309) were found to be protective factors. CONCLUSIONS: This study found that a higher proportion of phospholipids in intermediate-density lipoprotein and the various subfractions of low-density lipoprotein, including large LDL, medium LDL, and small LDL, is associated with a lower risk of developing diabetic kidney disease.

Aberrant expression of NEDD4L disrupts mitochondrial homeostasis by downregulating CaMKKß in diabetic kidney disease.

Disturbance in mitochondrial homeostasis within proximal tubules is a critical characteristic associated with diabetic kidney disease (DKD). CaMKKß/AMPK signaling plays an important role in regulating mitochondrial homeostasis. Despite the downregulation of CaMKKß in DKD pathology, the underlying mechanism remains elusive. The expression of NEDD4L, which is primarily localized to renal proximal tubules, is significantly upregulated in the renal tubules of mice with DKD. Coimmunoprecipitation (Co-IP) assays revealed a physical interaction between NEDD4L and CaMKKß. Moreover, deletion of NEDD4L under high glucose conditions prevented rapid CaMKKß protein degradation. In vitro studies revealed that the aberrant expression of NEDD4L negatively influences the protein stability of CaMKKß. This study also explored the role of NEDD4L in DKD by using AAV-shNedd4L in db/db mice. These findings confirmed that NEDD4L inhibition leads to a decrease in urine protein excretion, tubulointerstitial fibrosis, and oxidative stress, and mitochondrial dysfunction. Further in vitro studies demonstrated that si-Nedd4L suppressed mitochondrial fission and reactive oxygen species (ROS) production, effects antagonized by si-CaMKKß. In summary, the findings provided herein provide strong evidence that dysregulated NEDD4L disturbs mitochondrial homeostasis by negatively modulating CaMKKß in the context of DKD. This evidence underscores the potential of therapeutic interventions targeting NEDD4L and CaMKKß to safeguard renal tubular function in the management of DKD.