Metformin Protects Against Cisplatin-Induced Tubular Cell Apoptosis and Acute Kidney Injury via AMPKα-regulated Autophagy Induction.
ABSTRACT: Metformin, one of the most common prescriptions for patients with type 2 diabetes, is reported to protect the kidney from gentamicin-induced nephrotoxicity. However, the role and mechanisms for metformin in preventing cisplatin-induced nephrotoxicity remains largely unknown. In this study, a single intraperitoneal injection of cisplatin was employed to induce acute kidney injury (AKI) in CD1 mice. The mice exhibited severe kidney dysfunction and histological damage at day 2 after cisplatin injection. Pretreatment of metformin could markedly attenuate cisplatin-induced acute kidney injury, tubular cell apoptosis and inflammatory cell accumulation in the kidneys. Additionally, pretreatment of metformin could enhance both AMPKα phosphorylation and autophagy induction in the kidneys after cisplatin injection. In cultured NRK-52E cells, a rat kidney tubular cell line, metformin could stimulate AMPKα phosphorylation, induce autophagy and inhibit cisplatin-induced cell apoptosis. Blockade of either AMPKα activation or autophagy induction could largely abolish the protective effect of metformin in cisplatin-induced cell death. Together, this study demonstrated that metformin may protect against cisplatin-induced tubular cell apoptosis and AKI through stimulating AMPKα activation and autophagy induction in the tubular cells.
Project description:Aims: Cisplatin, an anticancer drug, always leads to nephrotoxicity by causing mitochondrial dysfunction. As a major mechanism for cellular self-degradation, autophagy has been proven to protect against cisplatin-induced acute kidney injury (AKI). Based on the activation of autophagy induced by trehalose, we aimed to investigate the nephroprotective effects of trehalose on cisplatin-induced AKI and its underlying mechanisms. Results: Due to the activation of autophagy, mitochondrial dysfunction (mitochondrial fragmentation, depolarization, reactive oxygen species (ROS), and reduced ATP generation) and apoptosis induced by cisplatin were markedly inhibited in trehalose-treated HK2 cells in vitro. Based on the transcriptional regulation role of transcription factor EB (TFEB) in autophagy and lysosome, we characterized trehalose-induced nuclear translocation of TFEB. Furthermore, consistent with trehalose treatment, overexpression of TFEB inhibited cell injury induced by cisplatin. However, the protective effects of trehalose were largely abrogated in tfeb-knockdown cells. In vivo, cisplatin injection resulted in severe kidney dysfunction and histological damage in mice. Trehalose administration activated TFEB-mediated autophagy, alleviated mitochondrial dysfunction and kidney injury in AKI mice. Innovation and conclusion: Our data suggest that trehalose treatment preserves mitochondria function via activation of TFEB-mediated autophagy and attenuates cisplatin-induced kidney injury.
Project description:Inappropriate activation of the renin-angiotensin system (RAS) contributes to many CKDs. However, the role of the RAS in modulating AKI requires elucidation, particularly because stimulating type 1 angiotensin II (AT1) receptors in the kidney or circulating inflammatory cells can have opposing effects on the generation of inflammatory mediators that underpin the pathogenesis of AKI. For example, TNF-? is a fundamental driver of cisplatin nephrotoxicity, and generation of TNF-? is suppressed or enhanced by AT1 receptor signaling in T lymphocytes or the distal nephron, respectively. In this study, cell tracking experiments with CD4-Cre mT/mG reporter mice revealed robust infiltration of T lymphocytes into the kidney after cisplatin injection. Notably, knockout of AT1 receptors on T lymphocytes exacerbated the severity of cisplatin-induced AKI and enhanced the cisplatin-induced increase in TNF-? levels locally within the kidney and in the systemic circulation. In contrast, knockout of AT1 receptors on kidney epithelial cells ameliorated the severity of AKI and suppressed local and systemic TNF-? production induced by cisplatin. Finally, disrupting TNF-? production specifically within the renal tubular epithelium attenuated the AKI and the increase in circulating TNF-? levels induced by cisplatin. These results illustrate discrepant tissue-specific effects of RAS stimulation on cisplatin nephrotoxicity and raise the concern that inflammatory mediators produced by renal parenchymal cells may influence the function of remote organs by altering systemic cytokine levels. Our findings suggest selective inhibition of AT1 receptors within the nephron as a promising intervention for protecting patients from cisplatin-induced nephrotoxicity.
Project description:Cisplatin (CP)-induced nephrotoxicity is widely accepted as a model for acute kidney injury (AKI). Although cisplatin-induced chronic kidney disease (CKD) in rodent has been reported, the role of phosphate in the cisplatin-induced CKD progression is not described. In this study, we gave a single peritoneal injection of CP followed by high (2%) phosphate diet for 20 weeks. High dose CP (20 mg/Kg) led to high mortality; whereas a lower dose (10 mg/Kg) resulted in a full spectrum of AKI with tubular necrosis, azotemia, and 0% mortality 7 days after CP injection. After consuming a high phosphate diet, mice developed CKD characterized by low creatinine clearance, interstitial fibrosis, hyperphosphatemia, high plasma PTH and FGF23, low plasma 1,25(OH)2 Vitamin D3 and αKlotho, and classic uremic cardiovasculopathy. The CP model was robust in demonstrating the effect of aging, sexual dimorphism, and dietary phosphate on AKI and also AKI-to-CKD progression. Finally, we used the CP-high phosphate model to examine previously validated methods of genetically manipulated high αKlotho and therapy using exogenous soluble αKlotho protein supplementation. In this CP CKD model, αKlotho mitigated CKD progression, improved mineral homeostasis, and ameliorated cardiovascular disease. Taken together, CP and high phosphate nephrotoxicity is a reproducible and technically very simple model for the study of AKI, AKI-to-CKD progression, extrarenal complications of CKD, and for evaluation of therapeutic efficacy.
Project description:Fatty acid-binding protein 4 (FABP4) has been confirmed to be involved in the pathogenesis of ischaemia/reperfusion- and rhabdomyolysis-induced acute kidney injury (AKI), and targeting inhibition of FABP4 might be a potential strategy for AKI. Cisplatin as a commonly used cancer chemotherapeutic drug possessed a dose-limited side effect of nephrotoxicity. However, whether FABP4 inhibition exerted a favourable renoprotection against cisplatin-induced AKI and the involved mechanisms remained unknown. In the study, cisplatin-injected mice developed severe AKI symptom as indicated by renal dysfunction and pathological changes, companied by the high expression of FABP4 in tubular epithelial cells. Selective inhibition of FABP4 by BMS309403 at 40 mg/kg/d for 3 days and genetic knockout of FABP4 significantly attenuated the serum creatinine, blood urea nitrogen level and renal tubular damage. Mechanistically, cisplatin injection induced the increased apoptosis and regulated the corresponding protein expression of BCL-2, BCL-XL, BAX, cleaved caspase 3 and caspase 12 in the injured kidney tissues. Cisplatin also triggered multiple signal mediators of endoplasmic reticulum (ER) stress including double-stranded RNA-activated protein kinase-like ER kinase, activating transcription factor-6 and inositol-requiring enzyme-1 pathway, as well as CHOP, GRP78 and p-JNK proteins in the kidneys. Oral administration of BMS309403 significantly reduced the number of renal TUNEL-positive apoptotic cells. Knockout of FABP4 and BMS309403 notably improved ER stress-related apoptotic responses. In summary, pharmacological and genetic inhibition of FABP4 modulated apoptosis via the inactivation of ER stress in the tubular epithelial cells of cisplatin-induced AKI.
Project description:The administration of cisplatin is limited due to its nephrotoxic side effects, and prevention of this nephrotoxicity of cisplatin is difficult. Mesenchymal stem cell (MSC)-derived exosomes have been implicated as a novel therapeutic approach for tissue injury. In this study, we demonstrated that the pretreatment of human umbilical cord MSC-derived exosomes (hucMSC-Ex) can prevent the development of cisplatin-induced renal toxicity by activation of autophagy in vitro and in vivo.In vitro, rat renal tubular epithelial (NRK-52E) cells were pre-incubated with exosomes from hucMSC or HFL1 (human lung fibroblast cells; as control) for 30 min, and 3-methyladenine (an autophagic inhibitor) and rapamycin (an autophagic inducer) for 1 h before cisplatin treatment for 8 h, respectively. Cells were harvested for apoptosis assay, enzyme-linked immunosorbent assay (ELISA), Western blot, and quantitative real-time polymerase chain reaction (qRT-PCR). In vivo, we constructed cisplatin-induced acute kidney injury rat models. Prior to treatment with cisplatin for 0.5 h, hucMSC-Ex or HFL1-Ex were injected into the kidneys via the renal capsule. 3-methyladenine and rapamycin were injected under the kidney capsule before hucMSC-Ex. All animals were sacrificed at 3 days after cisplatin injection. Renal function, Luminex assay, tubular apoptosis and proliferation, and autophagy response were evaluated.hucMSC-Ex inhibited cisplatin-induced mitochondrial apoptosis and secretion of inflammatory cytokines in renal tubular epithelial cells in vitro. hucMSC-Ex increased the expression of the autophagic marker protein LC3B and the autophagy-related genes ATG5 and ATG7 in NRK-52E cells. Rapamycin mimicked the effects of hucMSC-Ex in protecting against cisplatin-induced renal injury, while the effects were abrogated by the autophagy inhibitor 3-methyladenine in the animals.Our findings indicate that the activation of autophagy induced by hucMSC-Ex can effectively relieve the nephrotoxicity of cisplatin. Therefore, pre-treatment of hucMSC-Ex may be a new method to improve the therapeutic effect of cisplatin.
Project description:Ras homolog enriched in brain (Rheb1), a small GTPase, plays a crucial role in regulating cell growth, differentiation, and survival. However, the role and mechanisms for Rheb1 in tubular cell survival and acute kidney injury (AKI) remain unexplored. Here we found that Rheb1 signaling was activated in kidney tubule of AKI patients and cisplatin-treated mice. A mouse model of tubule-specific deletion of Rheb1 (Tubule-Rheb1-/-) was generated. Compared to control littermates, Tubule-Rheb1-/- mice were phenotypically normal within 2 months after birth but developed more severe kidney dysfunction, tubular cell death including apoptosis, necroptosis and ferroptosis, mitochondrial defect and less PGC-1? expression after cisplatin injection. In primary cultured tubular cells, Rheb1 ablation exacerbated cisplatin-induced cell death and mitochondrial defect. Furthermore, haploinsufficiency for Tsc1 in tubular cells led to Rheb1 activation and mitigated cisplatin-induced cell death, mitochondrial defect and AKI. Together, this study uncovers that Rheb1 may protect against cisplatin-induced tubular cell death and AKI through maintaining mitochondrial homeostasis.
Project description:Histone deacetylase inhibitors (HDACi) have therapeutic effects in models of various renal diseases including acute kidney injury (AKI); however, the underlying mechanism remains unclear. Here we demonstrate that two widely tested HDACi (suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA)) protect the kidneys in cisplatin-induced AKI by enhancing autophagy. In cultured renal proximal tubular cells, SAHA and TSA enhanced autophagy during cisplatin treatment. We further verified the protective effect of TSA against cisplatin-induced apoptosis in these cells. Notably, inhibition of autophagy by chloroquine or by autophagy gene 7 (Atg7) ablation diminished the protective effect of TSA. In mice, TSA increased autophagy in renal proximal tubules and protected against cisplatin-induced AKI. The in vivo effect of TSA was also abolished by chloroquine and by Atg7 knockout specifically from renal proximal tubules. Mechanistically, TSA stimulated AMPK and inactivated mTOR during cisplatin treatment of proximal tubule cells and kidneys in mice. Together, these results suggest that HDACi may protect kidneys by activating autophagy in proximal tubular cells.
Project description:Acute kidney injury (AKI) is a potentially fatal syndrome characterized by a rapid decline in kidney function caused by ischemic or toxic injury to renal tubular cells. The widely used chemotherapy drug cisplatin accumulates preferentially in the renal tubular cells and is a frequent cause of drug-induced AKI. During the development of AKI the quiescent tubular cells reenter the cell cycle. Strategies that block cell-cycle progression ameliorate kidney injury, possibly by averting cell division in the presence of extensive DNA damage. However, the early signaling events that lead to cell-cycle activation during AKI are not known. In the current study, using mouse models of cisplatin nephrotoxicity, we show that the G1/S-regulating cyclin-dependent kinase 4/6 (CDK4/6) pathway is activated in parallel with renal cell-cycle entry but before the development of AKI. Targeted inhibition of CDK4/6 pathway by small-molecule inhibitors palbociclib (PD-0332991) and ribociclib (LEE011) resulted in inhibition of cell-cycle progression, amelioration of kidney injury, and improved overall survival. Of additional significance, these compounds were found to be potent inhibitors of organic cation transporter 2 (OCT2), which contributes to the cellular accumulation of cisplatin and subsequent kidney injury. The unique cell-cycle and OCT2-targeting activities of palbociclib and LEE011, combined with their potential for clinical translation, support their further exploration as therapeutic candidates for prevention of AKI.
Project description:Cisplatin and its derivatives are widely used chemotherapeutic drugs for cancer treatment. However, they have debilitating side effects in normal tissues and induce ototoxicity, neurotoxicity, and nephrotoxicity. In kidneys, cisplatin preferentially accumulates in renal tubular cells causing tubular cell injury and death, resulting in acute kidney injury (AKI). Recent studies have suggested that DNA damage and the associated DNA damage response (DDR) are an important pathogenic mechanism of AKI following cisplatin treatment. Activation of DDR may lead to cell cycle arrest and DNA repair for cell survival or, in the presence of severe injury, kidney cell death. Modulation of DDR may provide novel renoprotective strategies for cancer patients undergoing cisplatin chemotherapy.
Project description:Acute kidney injury and nephrotoxicity are important clinical side effects of cisplatin. Thus, the mechanisms of this disease, and potential treatment options are important to understand in their entity. Here, we analyzed the proteome of cisplatin induced acute kidney injury in a mouse model. Functionally we found that calorie restriction was able to completely blunt Cisplatin induced AKI, and hypoxia ameliorated cCisplatin induced AKI. To investigate the mechanism for this in high throughput, we performed label-free single-shot proteomic analyses of these kidneys.Acute kidney injury and nephrotoxicity are important clinical side effects of cisplatin. Thus, the mechanisms of this disease, and potential treatment options are important to understand in their entity. Here, we analyzed the proteome of cisplatin induced acute kidney injury in a mouse model. Functionally we found that calorie restriction was able to completely blunt Cisplatin induced AKI, and hypoxia ameliorated cCisplatin induced AKI. To investigate the mechanism for this in high throughput, we performed label-free single-shot proteomic analyses of these kidneys.