Activation of Transcription Factor EB Alleviates Tubular Epithelial Cell Injury via Restoring Lysosomal Homeostasis in Diabetic Nephropathy.
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SUBMITTER: Wang S
PROVIDER: S-EPMC8786470 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:IntroductionKidney tubular epithelial injury is one of the key factors in the progression of diabetic nephropathy (DN). Wogonin is a kind of flavonoid, which has many pharmacological effects, such as anti-inflammation, anti-oxidation and anti-fibrosis. However, the effect of wogonin in renal tubular epithelial cells during DN is still unknown.Materials and methodsSTZ-induced diabetic mice were given doses of wogonin (10, 20, and 40 mg/kg) by intragastric administration for 16 weeks. The metabolic indexes from blood and urine and pathological damage of renal tubules in mice were evaluated. Human tubular epithelial cells (HK-2) were cultured in high glucose (HG) condition containing wogonin (2μM, 4μM, 8μM) for 24 h. Tubular epithelial cell inflammation and autophagic dysfunction both in vivo and in vitro were assessed by Western blot, qRT-PCR, IHC, and IF analyses.ResultsThe treatment of wogonin attenuated urinary albumin and histopathological damage in tubulointerstitium of diabetic mice. We also found that wogonin down-regulated the expression of pro-inflammatory cytokines and autophagic dysfunction in vivo and in vitro. Molecular docking and Cellular Thermal Shift Assay (CETSA) results revealed that mechanistically phosphoinositide 3-kinase (PI3K) was the target of wogonin. We then found that inhibiting PI3K eliminated the protective effect of wogonin. Wogonin regulated autophagy and inflammation via targeting PI3K, the important connection point of PI3K/Akt/NF-κB signaling pathway.ConclusionOur study is the first to demonstrate the novel role of wogonin in mitigating tubulointerstitial fibrosis and renal tubular cell injury via regulating PI3K/Akt/NF-κB signaling pathway-mediated autophagy and inflammation. Wogonin might be a latent remedial drug against tubular epithelial injury in DN by targeting PI3K.
| S-EPMC8291679 | biostudies-literature
Project description:Phosphatase and Tensin Homolog on chromosome Ten (PTEN) has emerged as a key protein that governs the response to kidney injury. Notably, renal adaptive repair is important for preventing acute kidney injury (AKI) to chronic kidney disease (CKD) transition. To test the role of PTEN in renal repair after acute injury, we constructed a mouse model that overexpresses PTEN in renal proximal tubular cells (RPTC) by crossing PTENfl-stop-fl mice with Ggt1-Cre mice. Mass spectrometry-based proteomics was performed after subjecting these mice to ischemia/reperfusion (I/R). We found that PTEN was downregulated in renal tubular cells in mice and cultured HK-2 cells subjected to renal maladaptive repair induced by I/R. Renal expression of PTEN negatively correlated with NGAL and fibrotic markers. RPTC-specific PTEN overexpression relieved I/R-induced maladaptive repair, as indicated by alleviative tubular cell damage, apoptosis, and subsequent renal fibrosis. Mass spectrometry analysis revealed that differentially expressed proteins in RPTC-specific PTEN overexpression mice subjected to I/R were significantly enriched in phagosome, PI3K/Akt, and HIF-1 signaling pathway and found significant upregulation of CHMP2A, an autophagy-related protein. PTEN deficiency downregulated CHMP2A and inhibited phagosome closure and autolysosome formation, which aggravated cell injury and apoptosis after I/R. PTEN overexpression had the opposite effect. Notably, the beneficial effect of PTEN overexpression on autophagy flux and cell damage was abolished when CHMP2A was silenced. Collectively, our study suggests that PTEN relieved renal maladaptive repair in terms of cell damage, apoptosis, and renal fibrosis by upregulating CHMP2A-mediated phagosome closure, suggesting that PTEN/CHMP2A may serve as a novel therapeutic target for the AKI to CKD transition.
| S-EPMC8599682 | biostudies-literature
Project description:Diabetic nephropathy is considered one of the most common microvascular complications of diabetes and the pathophysiology involves multiple factors. Progressive diabetic nephropathy is believed to be related to the structure and function of the tubular epithelial cells in the kidney. However, the role of lysine acetylation in lesions of the renal tubular epithelial cells arising from hyperglycemia is poorly understood. Consequently, in this study, we cultured mouse renal tubular epithelial cells in vitro under high glucose conditions and analyzed the acetylation levels of proteins by liquid chromatography-high-resolution mass spectrometry. We identified 48 upregulated proteins and downregulated 86 proteins. In addition, we identified 113 sites with higher acetylation levels and 374 sites with lower acetylation levels. Subcellular localization analysis showed that the majority of the acetylated proteins were located in the mitochondria (43.17%), nucleus (28.57%) and cytoplasm (16.19%). Enrichment analysis indicated that these acetylated proteins are primarily associated with oxidative phosphorylation, the citrate cycle (TCA cycle), metabolic pathways and carbon metabolism. In addition, we used the MCODE plug-in and the cytoHubba plug-in in Cytoscape software to analyze the PPI network and displayed the first four most compact MOCDEs and the top 10 hub genes from the differentially expressed proteins between global and acetylated proteomes. Finally, we extracted 37 conserved motifs from 4915 acetylated peptides. Collectively, this comprehensive analysis of the proteome reveals novel insights into the role of lysine acetylation in tubular epithelial cells and may make a valuable contribution towards the identification of the pathological mechanisms of diabetic nephropathy.
| S-EPMC8657754 | biostudies-literature
Project description:Silent information regulator 1 (Sirt1) is a deacetylase, which plays an important role in the occurrence and development of diabetic nephropathy (DN). Our previous study shows that Yin yang 1 (YY1), a widely expressed zinc finger DNA/RNA-binding transcription factor, is a novel regulator of renal fibrosis in diabetic nephropathy. Since the activity of YY1 is regulated via acetylation and deacetylation modification, this study aimed to explore whether Sirt1-induced deacetylation of YY1 mediated high glucose (HG)-induced renal tubular epithelial-mesenchymal transition (EMT) and renal fibrosis in vivo and in vitro. We first confirmed that Sirt1 expression level was significantly decreased in the kidney of db/db mice and in HG-treated HK-2 cells. Diabetes-induced Sirt1 reduction enhanced the level of YY1 acetylation and renal tubular EMT. Then, we manipulated Sirt1 expression in vivo and in vitro by injecting resveratrol (50 mg·kg-1·d-1. ip) to db/db mice for 2 weeks or application of SRT1720 (2.5 μM) in HG-treated HK-2 cells, we found that activation of Sirt1 reversed the renal tubular EMT and YY1 acetylation induced by HG condition. On the contrary, Sirt1 was knocked down in db/m mice or EX527 (1 μM) was added in HK-2 cells, we found that inhibition of Sirt1 exacerbated renal fibrosis in diabetic mice and enhanced level of YY1 acetylation in HK-2 cells. Furthermore, knockdown of YY1 inhibited the ameliorating effect of resveratrol on renal tubular EMT and renal fibrosis in db/db mice. In conclusion, this study demonstrates that Sirt1 plays an important role in renal tubular EMT of DN through mediating deacetylation of YY1.
| S-EPMC8027604 | biostudies-literature
Project description:Premature senescence is a key process in the progression of diabetic nephropathy (DN). Premature senescence of renal tubular epithelial cells (RTEC) in DN may result from the accumulation of damaged mitochondria. Mitophagy is the principal process that eliminates damaged mitochondria through PTEN-induced putative kinase 1 (PINK1)-mediated recruitment of optineurin (OPTN) to mitochondria. We aimed to examine the involvement of OPTN in mitophagy regulation of cellular senescence in RTEC in the context of DN. In vitro, the expression of senescence markers P16, P21, DcR2, SA-β-gal, SAHF, and insufficient mitophagic degradation marker (mitochondrial P62) in mouse RTECs increased after culture in 30 mM high-glucose (HG) conditions for 48 h. Mitochondrial fission/mitophagy inhibitor Mdivi-1 significantly enhanced RTEC senescence under HG conditions, whereas autophagy/mitophagy agonist Torin1 inhibited cell senescence. MitoTempo inhibited HG-induced mitochondrial reactive oxygen species and cell senescence with or without Mdivi-1. The expression of PINK1 and OPTN, two regulatory factors for mitophagosome formation, decreased significantly after HG stimulation. Overexpression of PINK1 did not enhance mitophagosome formation under HG conditions. OPTN silencing significantly inhibited HG-induced mitophagosome formation, and overexpression of OPTN relieved cellular senescence through promoting mitophagy. In clinical specimens, renal OPTN expression was gradually decreased with increased tubulointerstitial injury scores. OPTN-positive renal tubular cells did not express senescence marker P16. OPTN expression also negatively correlated with serum creatinine levels, and positively correlated with eGFR. Thus, OPTN-mediated mitophagy plays a crucial regulatory role in HG-induced RTEC senescence in DN. OPTN may, therefore, be a potential antisenescence factor in DN.
| S-EPMC5833650 | biostudies-literature
Project description:Diabetic nephropathy (DN), a leading cause of end-stage renal disease, remains a formidable challenge in diabetes management due to the complex nature of its pathogenesis, particularly the epithelial-mesenchymal transition (EMT) process. Our innovative study leverages network pharmacology to explore the therapeutic potentials of Myricetin, a natural flavonoid, focusing on its effects against NOX4, a critical mediator in DN progression. This investigation marks a pioneering approach by integrating network pharmacology to predict and elucidate the inhibitory relationship between Myricetin and NOX4. Utilizing a high-fat diet/streptozotocin (HFD/STZ) induced DN mouse model, we delved into the effects of Myricetin on renal EMT processes. Through network pharmacology analyses coupled with molecular docking studies, we identified and confirmed Myricetin's binding efficacy to NOX4. Extensive in vitro and in vivo experiments further established Myricetin's significant impact on mitigating EMT by modulating the NOX4-NF-κB-Snail signaling pathway. Results from our research demonstrated notable improvements in renal function and reductions in tissue fibrosis among treated HFD/STZ mice. By curtailing NOX4 expression, Myricetin effectively reduced reactive oxygen species (ROS) production, thereby inhibiting NF-κB activation and subsequent Snail expression, crucial steps in the EMT pathway. Supported by both theoretical predictions and empirical validations, this study unveils the mechanism underlying Myricetin's modulation of EMT in DN through disrupting the NOX4-NF-κB-Snail axis. These findings not only contribute a new therapeutic avenue for DN treatment but also underscore the utility of network pharmacology in advancing drug discovery processes.
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Project description:Renal tubular epithelial cells (RTECs) are one of the most mitochondria-rich cell types, and are thus vulnerable to mitochondrial dysregulation, which is defined as a pivotal event in tubular damage in diabetic nephropathy (DN). However, the underlying mechanisms remain largely unknown. Here, we investigated the role and mechanisms of tumor necrosis factor alpha-induced protein 8-like 1 (TNFAIP8L1/TIPE1) in high glucose (HG)-induced mitochondrial dysfunction in RTECs and DN progression. TIPE1 expression was predominantly upregulated in RTECs in patients with DN and mice with streptozotocin (STZ)-induced DN. Conditional knockout of Tipe1 in RTECs significantly decreased the urine protein creatinine ratio, renal tubular damage, epithelial-mesenchymal transition, and interstitial fibrosis in STZ-induced mice. RNA sequencing revealed that citrate cycle-related genes were positively enriched in the renal tissues of RTEC-specific Tipe1 knockout mice. Tipe1 deficiency upregulated ATP levels, mitochondrial membrane potential, and respiration rate, but downregulated mitochondrial ROS levels in RTECs. Furthermore, Tipe1 ablation led to enhanced mitophagy in RTECs, indicative of increased LC3II, PINK1, and Parkin expression, but decreased p62 expression in mitochondria. Mechanistically, mass spectrometry screening and co-immunoprecipitation assays revealed the interaction of TIPE1 with prohibitin 2 (PHB2), a crucial mitophagy receptor. Intriguingly, TIPE1 promoted the ubiquitination and proteasomal degradation of PHB2. Subsequently, PHB2 knockdown almost abrogated the improvement of Tipe1 loss on HG-induced tubular cell mitophagy and damage. Thus, TIPE1 disrupts mitochondrial homeostasis in RTECs and promotes tubular damage by destabilizing PHB2 under HG conditions. Hence, TIPE1 may act as a potential therapeutic target to prevent DN progression.
| S-EPMC8844679 | biostudies-literature
Project description:Macroautophagy/autophagy dysregulation has been noted in diabetic nephropathy; however, the regulatory mechanisms controlling this process remain unclear. In this study, we showed that SMAD3 (SMAD family member 3), the key effector of TGFB (transforming growth factor beta)-SMAD signaling, induces lysosome depletion via the inhibition of TFEB-dependent lysosome biogenesis. The pharmacological inhibition or genetic deletion of SMAD3 restored lysosome biogenesis activity by alleviating the suppression of TFEB, thereby protecting lysosomes from depletion and improving autophagic flux in renal tubular epithelial cells in diabetic nephropathy. Mechanistically, we found that SMAD3 directly binds to the 3'-UTR of TFEB and inhibits its transcription. Silencing TFEB suppressed lysosome biogenesis and resulted in a loss of the protective effects of SMAD3 inactivation on lysosome depletion under diabetic conditions. In conclusion, SMAD3 promotes lysosome depletion via the inhibition of TFEB-dependent lysosome biogenesis; this may be an important mechanism underlying autophagy dysregulation in the progression of diabetic nephropathy.Abbreviations: AGEs: advanced glycation end products; ATP6V1H: ATPase H+ transporting V1 subunit H; CTSB: cathepsin B; ChIP: chromatin immunoprecipitation; Co-BSA: control bovine serum albumin; DN: diabetic nephropathy; ELISA: enzyme-linked immunosorbent assay; FN1: fibronectin 1; HAVCR1/TIM1/KIM-1: hepatitis A virus cellular receptor 1; LAMP1: lysosomal associated membrane protein 1; LMP: lysosome membrane permeabilization; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; NC: negative control; SIS3: specific inhibitor of SMAD3; SMAD3: SMAD family member 3; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; TECs: tubular epithelial cells; TFEB: transcription factor EB; TGFB1: transforming growth factor beta 1; TGFBR1: transforming growth factor beta receptor 1; UTR: untranslated region; VPS11: VPS11 core subunit of CORVET and HOPS complexes.
| S-EPMC8496726 | biostudies-literature
Project description:Diabetic nephropathy (DN) is the leading cause of end-stage renal disease (ESRD), and renal tubular cell dysfunction contributes to the pathogenesis of DN. Soluble epoxide hydrolase (sEH) is an enzyme that can hydrolyze epoxyeicosatrienoic acids (EETs) and other epoxy fatty acids (EpFAs) into the less biologically active metabolites. Inhibition of sEH has multiple beneficial effects on renal function, however, the exact role of sEH in hyperglycemia-induced dysfunction of tubular cells is still not fully elucidated. In the present study, we showed that human proximal tubular epithelial (HK-2) cells revealed an upregulation of sEH expression accompanied by the impairment of autophagic flux, mitochondrial dysfunction, ubiquitinated protein accumulation and enhanced endoplasmic reticulum (ER) stress after high glucose (HG) treatment. Furthermore, dysfunctional mitochondria accumulated in the cytoplasm, which resulted in excessive reactive oxygen species (ROS) generation, Bax translocation, cytochrome c release, and apoptosis. However, t-AUCB, an inhibitor of sEH, partially reversed these negative outcomes. Moreover, we also observed increased sEH expression, impaired autophagy flux, mitochondrial dysfunction and enhanced ER stress in the renal proximal tubular cells of db/db diabetic mice. Notably, inhibition of sEH by treatment with t-AUCB attenuated renal injury and partially restored autophagic flux, improved mitochondrial function, and reduced ROS generation and ER stress in the kidneys of db/db mice. Taken together, these results suggest that inhibition of sEH by t-AUCB plays a protective role in hyperglycemia-induced proximal tubular injury and that the potential mechanism of t-AUCB-mediated protective autophagy is involved in modulating mitochondrial function and ER stress. Thus, we provide new evidence linking sEH to the autophagic response during proximal tubular injury in the pathogenesis of DN and suggest that inhibition of sEH can be considered a potential therapeutic strategy for the amelioration of DN.
| S-EPMC7242354 | biostudies-literature
Project description:Rap1b ameliorates high glucose (HG)-induced mitochondrial dysfunction in tubular cells. However, its role and precise mechanism in diabetic nephropathy (DN) in vivo remain unclear. We hypothesize that Rap1 plays a protective role in tubular damage of DN by modulating primarily the mitochondria-derived oxidative stress. The role and precise mechanisms of Rap1b on mitochondrial dysfunction and of tubular cells in DN were examined in rats with streptozotocin (STZ)-induced diabetes that have Rap1b gene transfer using an ultrasound microbubble-mediated technique as well as in renal proximal epithelial tubular cell line (HK-2) exposed to HG ambiance. The results showed that Rap1b expression decreased significantly in tubules of renal biopsies from patients with DN. Overexpression of a constitutively active Rap1b G12V notably ameliorated renal tubular mitochondrial dysfunction, oxidative stress, and apoptosis in the kidneys of STZ-induced rats, which was accompanied with increased expression of transcription factor C/EBP-? and PGC-1?. Furthermore, Rap1b G12V also decreased phosphorylation of Drp-1, a key mitochondrial fission protein, while boosting the expression of genes related to mitochondrial biogenesis and antioxidants in HK-2 cells induced by HG. These effects were imitated by transfection with C/EBP-? or PGC-1? short interfering RNA. In addition, Rap1b could modulate C/EBP-? binding to the endogenous PGC-1? promoter and the interaction between PGC-1? and catalase or mitochondrial superoxide dismutase, indicating that Rap1b ameliorates tubular injury and slows the progression of DN by modulation of mitochondrial dysfunction via C/EBP-?-PGC-1? signaling.
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