Kidney-specific overexpression of Sirt1 protects against acute kidney injury by retaining peroxisome function.
ABSTRACT: Sirt1, a NAD-dependent protein deacetylase, is reported to regulate intracellular metabolism and attenuate reactive oxidative species (ROS)-induced apoptosis leading to longevity and acute stress resistance. We created transgenic (TG) mice with kidney-specific overexpression of Sirt1 using the promoter sodium-phosphate cotransporter IIa (Npt2) driven specifically in proximal tubules and investigated the kidney-specific role of Sirt1 in the protection against acute kidney injury (AKI). We also elucidated the role of number or function of peroxisome and mitochondria in mediating the mechanisms for renal protective effects of Sirt1 in AKI. Cisplatin-induced AKI decreased the number and function of peroxisomes as well as mitochondria and led to increased local levels of ROS production and renal tubular apoptotic cells. TG mice treated with cisplatin mitigated AKI, local ROS, and renal tubular apoptotic tubular cells. Consistent with these results, TG mice treated with cisplatin also exhibited recovery of peroxisome number and function, as well as rescued mitochondrial function; however, mitochondrial number was not recovered. Immunoelectron microscopic findings consistently demonstrated that the decrease in peroxisome number by cisplatin in wild type mice was restored in transgenic mice. In HK-2 cells, a cultured proximal tubule cell line, overexpression of Sirt1 rescued the cisplatin-induced cell apoptosis through the restoration of peroxisome number, although the mitochondria number was not restored. These results indicate that Sirt1 overexpression in proximal tubules rescues cisplatin-induced AKI by maintaining peroxisomes number and function, concomitant up-regulation of catalase, and elimination of renal ROS levels. Renal Sirt1 can be a potential therapeutic target for the treatment of AKI.
Project description:Acute kidney injury (AKI) is a common complication in cancer patients. Kidney function is closely related to patients' quality of life and tumor prognosis. Cisplatin is a highly effective anti-tumor drug. However, the use of cisplatin is limited by its nephrotoxicity. It has been reported that FGF21 has a renal-protective function, but the mechanisms by which it does so remain unclear. In this study, we show that the expression of FGF21 is significantly upregulated in both <i>in vitro</i> and <i>in vivo</i> cisplatin-induced AKI models. Administration of recombinant FGF21 to cisplatin-induced AKI mice resulted in significantly decreased blood urea nitrogen (BUN) and serum creatinine levels, as well as significantly reduced protein levels of kidney injury molecule-1 (TIM-1), C-caspase 3, and Bax. H&E-stained kidney sections from cisplatin-induced AKI mice treated with recombinant FGF21 showed a relatively normal renal tissue structure, a reduced number of necrotic sites and vacuolar changes, and decreased casts, suggesting alleviated renal tubular injury. Experiments with an AKI cell model (cisplatin-treated HK-2 cells) yielded similar results as the mouse model; recombinant FGF21 significantly downregulated protein expression levels of TIM-1, C-caspase 3, and Bax. Furthermore, administration of recombinant FGF21 to cisplatin-treated AKI models significantly increased SIRT1 expression, and the beneficial effects of FGF21 on kidney injury were reversed by <i>SIRT1</i> knockdown. Collectively, our results suggest that SIRT1 mediates the protective effect of FGF21 on cisplatin-induced kidney injury.
Project description:Overexpression of myo-inositol oxygenase (MIOX), a proximal tubular enzyme, exacerbates cellular redox injury in acute kidney injury (AKI). Ferroptosis, a newly coined term associated with lipid hydroperoxidation, plays a critical role in the pathogenesis of AKI. Whether or not MIOX exacerbates tubular damage by accelerating ferroptosis in cisplatin-induced AKI remains elusive. Cisplatin-treated HK-2 cells exhibited notable cell death, which was reduced by ferroptosis inhibitors. Also, alterations in various ferroptosis metabolic sensors, including lipid hydroperoxidation, glutathione peroxidase 4 (GPX4) activity, NADPH and reduced glutathione (GSH) levels, and ferritinophagy, were observed. These perturbations were accentuated by MIOX overexpression, while ameliorated by MIOX knockdown. Likewise, cisplatin-treated CD1 mice exhibited tubular damage and derangement of renal physiological parameters, which were alleviated by ferrostatin-1, a ferroptosis inhibitor. To investigate the relevance of MIOX to ferroptosis, WT mice, MIOX-overexpressing transgenic (MIOX-Tg) mice, and MIOX-KO mice were subjected to cisplatin treatment. In comparison with cisplatin-treated WT mice, cisplatin-treated MIOX-Tg mice had more severe renal pathological changes and perturbations in ferroptosis metabolic sensors, which were minimal in cisplatin-treated MIOX-KO mice. In conclusion, these findings indicate that ferroptosis, an integral process in the pathogenesis of cisplatin-induced AKI, is modulated by the expression profile of MIOX.
Project description:<h4>Background</h4>Uncoupling protein 1 (UCP1) is predominantly found in brown adipose tissue mitochondria, and mediates energy dissipation to generate heat rather than ATP via functional mitochondrial uncoupling. However, little is known about its expression and function in kidney.<h4>Methods</h4>We carried out a mRNA microarray analysis in mice kidneys with ischemia reperfusion (IR) injury. The most dramatically downregulated gene UCP1 after IR was identified, and its role in generation of mitochondrial reactive oxygen species (ROS) and oxidative stress injury was assessed both in vitro and in vivo. Genetic deletion of UCP1 was used to investigate the effects of UCP1 on ischemia or cisplatin-indued acute kidney injury (AKI) in mice.<h4>Findings</h4>UCP1 was located in renal tubular epithelial cells in kidney and downregulated in a time-dependent manner during renal IR. Deletion of UCP1 increased oxidative stress in kidneys and aggravated ischemia or cisplatin induced AKI in mice.Viral-based overexpression of UCP1 reduced mitochondrial ROS generation and apoptosis in hypoxia-treated tubular epithelial cells. Furthermore, UCP1 expression was regulated by peroxisome proliferator-activator receptor (PPAR) ? in kidneys during renal IR. Overexpression of PPAR-? resembled UCP1-overexpression phenotype in vitro. Treatment with PPAR-? agonist could induce UCP1 upregulation and provide protective effect against renal IR injury in UCP1<sup>+/+</sup>mice, but not in UCP1<sup>-/-</sup>mice.<h4>Interpretation</h4>UCP1 protects against AKI likely by suppressing oxidative stress, and activation of UCP1 represents a potential therapeutic strategy for AKI. FUND: National Natural Science Foundation of China grants, Science and Technology Commission of Shanghai.
Project description:Salusin-? is abundantly expressed in many organs and tissues including heart, blood vessels, brain and kidneys. Recent studies have identified salusin-? as a bioactive peptide that contributes to various diseases, such as atherosclerosis, hypertension, diabetes and metabolic syndrome. However, the role of salusin-? in the pathogenesis of acute kidney injury (AKI) is largely unclear. In the present study, we investigated the roles of salusin-? in cisplatin or lipopolysaccharide (LPS)-induced renal injury. Herein, we found that salusin-? expression was upregulated in both renal tubular cells and kidney tissues induced by both cisplatin and LPS. In vitro, silencing of salusin-? diminished, whereas overexpression of salusin-? exaggerated the increased PKC phosphorylation, oxidative stress, histone ?H2AX expression, p53 activation and apoptosis in either cisplatin or LPS-challenged renal tubular cells. More importantly, salusin-? overexpression-induced tubular cell apoptosis were abolished by using the PKC inhibitor Go 6976, reactive oxygen species (ROS) scavenger NAC, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor apocynin (Apo) or p53 inhibitor Pifithrin-?. In animals, blockade of salusin-? alleviated PKC phosphorylation, ROS accumulation, DNA damage, and p53 activation as well as renal dysfunction in mice after administration of cisplatin or LPS. Taken together, these results suggest that overexpressed salusin-? is deleterious in AKI by activation of the PKC/ROS signaling pathway, thereby priming renal tubular cells for apoptosis and death.
Project description:Resveratrol (Res) is a multi-functional polyphenol compound that has protective functions in acute kidney diseases. Here, we examined whether the resveratrol could ameliorate post-contrast acute kidney injury (PC-AKI) following diabetic nephropathy (DN), and explored any underlying mechanism(s) in vivo and in vitro. Twenty-four rabbits with DN were randomly divided into four groups: control (Cont), resveratrol (Res), iohexol (PC-AKI), and resveratrol plus iohexol (Res+PC-AKI) groups. Functional magnetic resonance imaging, renal histology, blood and urinary biomarkers, silent information regulator l (SIRT1), peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1?), hypoxia-inducible transcription factor-1? (HIF-1?), and apoptosis-associated protein expression were assessed ex vivo. For in vitro experiments, renal tubular epithelial (HK-2) cells subjected to high glucose conditions were treated with resveratrol, Ex527, an SIRT1 inhibitor, or 2-methoxyestradiol (2-MeOE2), HIF-1? inhibitor, before treatment with iohexol. With regard to the rabbit model of acute renal injury in DN, compared to the PC-AKI group, the Res+PC-AKI group showed decreased levels of cystatin C and urinary neutrophil gelatinase-associated lipocalin, increased pure molecular diffusion (D) and the fraction of water flowing in capillaries (f), a decreased apparent relaxation rate (R2*), renal injury score and apoptosis rate, increased protein expression levels of SIRT1 and PGC-1?, and decreased levels of HIF-1? and apoptosis-associated protein. In addition, iohexol decreased HK-2 cell survival and increased the cell apoptosis rate; results were reversed after treating cells with resveratrol. Resveratrol reduced renal hypoxia, mitochondrial dysfunction and renal tubular cell apoptosis by activating SIRT1-PGC-1?-HIF-1? signaling pathways in PC-AKI with DN.
Project description:Renal ischemia-reperfusion (I/R) can induce oxidative stress and injury via the generation of reactive oxygen species (ROS). Renal proximal tubular cells are susceptible to oxidative stress, and the dysregulation of renal proximal tubular cellular homeostasis can damage cells via apoptotic pathways. A recent study showed that the generation of ROS can increase perilipin 2 (Plin2) expression in HepG2 cells. Some evidence has also demonstrated the association between Plin2 expression and renal tumors. However, the underlying mechanism of Plin2 in I/R-induced acute kidney injury (AKI) remains elusive. Here, using a mouse model of I/R-induced AKI, we found that ROS generation was increased and the expression of Plin2 was significantly upregulated. An in vitro study further revealed that the expression of Plin2, and the generation of ROS were significantly upregulated in primary tubular cells treated with hydrogen peroxide. Accordingly, Plin2 knockdown decreased apoptosis in renal proximal tubular epithelial cells treated with hydrogen peroxide, which depended on the activation of peroxisome proliferator-activated receptor <i>α</i> (PPAR<i>α</i>). Overall, the present study demonstrated that Plin2 is involved in AKI; knockdown of this marker might limit apoptosis via the activation of PPAR<i>α</i>. Consequently, the downregulation of Plin2 could be a novel therapeutic strategy for AKI.
Project description:Demethylase Tet2 plays a vital role in the immune response. Acute kidney injury (AKI) initiation and maintenance phases are marked by inflammatory responses and leukocyte recruitment in endothelial and tubular cell injury processes. However, the role of Tet2 in AKI is poorly defined. Our study determined the degree of renal tissue damage associated with Tet2 gene expression levels in a cisplatin-induced AKI mice model. Tet2-knockout (KO) mice with cisplatin treatment experienced severe tubular necrosis and dilatation, inflammation, and AKI markers' expression levels than the wild-type mice. In addition, the administration of Tet2 plasmid protected Tet2-KO mice from cisplatin-induced nephrotoxicity, but not Tet2-catalytic-dead mutant. Tet2 KO was associated with a change in metabolic pathways like retinol, arachidonic acid, linolenic acid metabolism, and PPAR signaling pathway in the cisplatin-induced mice model. Tet2 expression is also downregulated in other AKI mice models and clinical samples. Thus, our results indicate that Tet2 has a renal protective effect during AKI by regulating metabolic and inflammatory responses through the PPAR signaling pathway.
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: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:Acute kidney injury (AKI) is a public health concern with an annual mortality rate that exceeds those of breast and prostate cancer, heart failure, and diabetes combined. Oxidative stress and mitochondrial damage are drivers of AKI-associated pathology; however, the pathways that mediate these events are poorly defined. Here, using a murine cisplatin-induced AKI model, we determined that both oxidative stress and mitochondrial damage are associated with reduced levels of renal sirtuin 3 (SIRT3). Treatment with the AMPK agonist AICAR or the antioxidant agent acetyl-l-carnitine (ALCAR) restored SIRT3 expression and activity, improved renal function, and decreased tubular injury in WT animals, but had no effect in Sirt3-/- mice. Moreover, Sirt3-deficient mice given cisplatin experienced more severe AKI than WT animals and died, and neither AICAR nor ALCAR treatment prevented death in Sirt3-/- AKI mice. In cultured human tubular cells, cisplatin reduced SIRT3, resulting in mitochondrial fragmentation, while restoration of SIRT3 with AICAR and ALCAR improved cisplatin-induced mitochondrial dysfunction. Together, our results indicate that SIRT3 is protective against AKI and suggest that enhancing SIRT3 to improve mitochondrial dynamics has potential as a strategy for improving outcomes of renal injury.