Massive intravascular hemolysis is associated with acute kidney injury (AKI). Nuclear factor erythroid-2-related factor 2 (Nrf2) plays a central role in the defense against oxidative stress by ...activating the expression of antioxidant proteins. We investigated the role of Nrf2 in intravascular hemolysis and whether Nrf2 activation protected against hemoglobin (Hb)/heme-mediated renal damage
and
. We observed renal Nrf2 activation in human hemolysis and in an experimental model of intravascular hemolysis promoted by phenylhydrazine intraperitoneal injection. In wild-type mice, Hb/heme released from intravascular hemolysis promoted AKI, resulting in decreased renal function, enhanced expression of tubular injury markers (KIM-1 and NGAL), oxidative and endoplasmic reticulum stress (ER), and cell death. These features were more severe in Nrf2-deficient mice, which showed decreased expression of Nrf2-related antioxidant enzymes, including heme oxygenase 1 (HO-1) and ferritin. Nrf2 activation with sulforaphane protected against Hb toxicity in mice and cultured tubular epithelial cells, ameliorating renal function and kidney injury and reducing cell stress and death. Nrf2 genotype or sulforaphane treatment did not influence the severity of hemolysis. In conclusion, our study identifies Nrf2 as a key molecule involved in protection against renal damage associated with hemolysis and opens novel therapeutic approaches to prevent renal damage in patients with severe hemolytic crisis. These findings provide new insights into novel aspects of Hb-mediated renal toxicity and may have important therapeutic implications for intravascular hemolysis-related diseases.
In chronic kidney disease patients, high phosphate (HP) levels are associated with cardiovascular disease, the major cause of morbidity and mortality. Since serum phosphate has been independently ...correlated with inflammation, the present study aimed to investigate an independent direct effect of HP as a pro-inflammatory factor in VSMCs. A possible modulatory effect of vitamin D (VitD) was also investigated. The study was performed in an
model of human aortic smooth muscle cells (HASMCs). Incubation of cells in an HP (3.3 mM) medium caused an increased expression of the pro-inflammatory mediators intercellular adhesion molecule 1 (ICAM-1), interleukins (ILs) IL-1β, IL-6, IL-8 and tumour necrosis factor α (TNF-α) (not corroborated at the protein levels for ICAM-1), as well as an increase in reactive oxygen/nitrogen species (ROS/RNS) production. This was accompanied by the activation of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) signalling as demonstrated by the increase in the nuclear translocation of nuclear factor κ-light-chain-enhancer of activated B cells protein 65 (p65-NF-κΒ) assessed by Western blotting and confocal microscopy. Since all these events were attenuated by an antioxidant pre-incubation with the radical scavenger Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP), it is suggested that the inflammatory response is upstream mediated by the ROS/RNS-induced activation of NF-κΒ. Addition of paricalcitol (PC) 3·10
M to cells in HP prevented the phosphate induced ROS/RNS increase, the activation of NF-κΒ and the cytokine up-regulation. A bimodal effect was observed, however, for different calcitriol (CTR) concentrations, 10
and 10
M attenuated but 10
M stimulated this phosphate induced pro-oxidative and pro-inflammatory response. Therefore, these findings provide novel mechanisms whereby HP may directly favour vascular dysfunctions and new insights into the protective effects exerted by VitD derivatives.
Toll-Like Receptors in Acute Kidney Injury Vázquez-Carballo, Cristina; Guerrero-Hue, Melania; García-Caballero, Cristina ...
International journal of molecular sciences,
01/2021, Letnik:
22, Številka:
2
Journal Article
Recenzirano
Odprti dostop
Acute kidney injury (AKI) is an important health problem, affecting 13.3 million individuals/year. It is associated with increased mortality, mainly in low- and middle-income countries, where renal ...replacement therapy is limited. Moreover, survivors show adverse long-term outcomes, including increased risk of developing recurrent AKI bouts, cardiovascular events, and chronic kidney disease. However, there are no specific treatments to decrease the adverse consequences of AKI. Epidemiological and preclinical studies show the pathological role of inflammation in AKI, not only at the acute phase but also in the progression to chronic kidney disease. Toll-like receptors (TLRs) are key regulators of the inflammatory response and have been associated to many cellular processes activated during AKI. For that reason, a number of anti-inflammatory agents targeting TLRs have been analyzed in preclinical studies to decrease renal damage during AKI. In this review, we updated recent knowledge about the role of TLRs, mainly TLR4, in the initiation and development of AKI as well as novel compounds targeting these molecules to diminish kidney injury associated to this pathological condition.
Transforming growth factor-β (TGF-β) is a key cytokine during differentiation of mesenchymal stem cells (MSC) into vascular smooth muscle cells (VSMC). High phosphate induces a phenotypic ...transformation of vascular smooth muscle cells (VSMC) into osteogenic-like cells. This study was aimed to evaluate signaling pathways involved during VSMC differentiation of MSC in presence or not of high phosphate.
Our results showed that TGF-β induced nuclear translocation of Smad3 as well as the expression of vascular smooth muscle markers, such as smooth muscle alpha actin, SM22α, myocardin, and smooth muscle-myosin heavy chain. The addition of high phosphate to MSC promoted nuclear translocation of Smad1/5/8 and the activation of canonical Wnt/β-catenin in addition to an increase in BMP-2 expression, calcium deposition and alkaline phosphatase activity. The administration of TGF-β to MSC treated with high phosphate abolished all these effects by inhibiting canonical Wnt, BMP and TGF-β pathways. A similar outcome was observed in high phosphate-treated cells after the inhibition of canonical Wnt signaling with Dkk-1. Conversely, addition of both Wnt/β-catenin activators CHIR98014 and lithium chloride enhanced the effect of high phosphate on BMP-2, calcium deposition and alkaline phosphatase activity.
Full VSMC differentiation induced by TGF-β may not be achieved when extracellular phosphate levels are high. Moreover, TGF-β prevents high phosphate-induced osteogenesis by decreasing the nuclear translocation of Smad 1/5/8 and avoiding the activation of Wnt/β-catenin pathway.
Periodontitis is a complex pathology characterized by the loss of alveolar bone. The causes and the mechanisms that promote this bone resorption still remain unknown. The knowledge of the critical ...regulators involved in the alteration of alveolar bone homeostasis is of great importance for developing molecular therapies. Procaine is an anesthetic drug with demethylant properties, mainly used by dentists in oral surgeries. The inhibitor role of Wnt signaling of procaine was described in vitro in colon cancer cells.
In this work we evaluated the role of procaine (1 uM) in osteo/odontogenesis of rat bone marrow mesenchymal stem cells. Similarly, the mechanisms whereby procaine achieves these effects were also studied.
Procaine administration led to a drastic decrease of calcium content, alkaline phosphatase activity, alizarin red staining and an increase in the expression of Matrix Gla Protein. With respect to osteo/odontogenic markers, procaine decreased early and mature osteo/odontogenic markers. In parallel, procaine inhibited canonical Wnt/β-catenin pathway, observing a loss of nuclear β-catenin, a decrease in Lrp5 and Frizzled 3, a significant increase of sclerostin and Gsk3β and an increase of phosphorylated β-catenin. The combination of osteo/odontogenic stimuli and Lithium Chloride decreased mRNA expression of Gsk3β, recovered by Procaine. Furthermore it was proved that Procaine alone dose dependently increases the expression of Gsk3β and β-catenin phosphorylation. These effects of procaine were also observed on mature osteoblast. Interestingly, at this concentration of procaine no demethylant effects were observed.
Our results demonstrated that procaine administration drastically reduced the mineralization and osteo/odontogenesis of bone marrow mesenchymal stem cells inhibiting Wnt/β-catenin pathway through the increase of Gsk3β expression and β-catenin phosphorylation.
Wnt/β-catenin pathway controls biochemical processes related to cell differentiation. In committed cells the alteration of this pathway has been associated with tumors as hepatocellular carcinoma or ...hepatoblastoma. The present study evaluated the role of Wnt/β-catenin activation during human mesenchymal stem cells differentiation into hepatocytes. The differentiation to hepatocytes was achieved by the addition of two different conditioned media. In one of them, β-catenin nuclear translocation, up-regulation of genes related to the Wnt/β-catenin pathway, such as Lrp5 and Fzd3, as well as the oncogenes c-myc and p53 were observed. While in the other protocol there was a Wnt/β-catenin inactivation. Hepatocytes with nuclear translocation of β-catenin also had abnormal cellular proliferation, and expressed membrane proteins involved in hepatocellular carcinoma, metastatic behavior and cancer stem cells. Further, these cells had also increased auto-renewal capability as shown in spheroids formation assay. Comparison of both differentiation protocols by 2D-DIGE proteomic analysis revealed differential expression of 11 proteins with altered expression in hepatocellular carcinoma. Cathepsin B and D, adenine phosphoribosyltransferase, triosephosphate isomerase, inorganic pyrophosphatase, peptidyl-prolyl cis-trans isomerase A or lactate dehydrogenase β-chain were up-regulated only with the protocol associated with Wnt signaling activation while other proteins involved in tumor suppression, such as transgelin or tropomyosin β-chain were down-regulated in this protocol. In conclusion, our results suggest that activation of the Wnt/β-catenin pathway during human mesenchymal stem cells differentiation into hepatocytes is associated with a tumoral phenotype.
Parallel to the safety of liver resections, new chemotherapy drugs have emerged for the control of liver metastases. However, there is unclear evidence about the combination of intensive BVZ-therapy ...and extended resections. The main aim was to analyse the impact of Bevacizumab (BVZ) in terms of liver safety and tolerability in two experimental models: a basal-toxicity situation and after major hepatectomy.
Eighty male-Wistar rats were grouped as toxicity analysis (sham-operated rats-OS-) and regeneration after- surgery analysis (hepatectomy rats-H-). Eight further subgroups were created according to sacrifice (6- hours-6h- or 24-hours-24h-) and dose (μg) of BVZ (none, 100, 200, 400). Several measurements were performed, including biochemical serum samples, histopathological analysis, cytokines (IL-6, TNF-α, TGF-β), oxidative-stress (GSH/GSSG, ATP), lipid-peroxidation (TBARS) and epidermal and vascular endothelium growth-factors (EGF and VEGF).
In the toxicity analysis, safe results with BVZ were observed, with no significant differences among the groups. A trend towards a lower oxidative status was observed in the OS 6 h-100, -200 and -400 versus the OS 6 h-none group. Similar results were observed in the hepatectomy model, with stable oxidative-stress-index and IL-6, TNF- α, and TGF- β levels. Despite higher lipid peroxidation status, overall regeneration was preserved. As expected, VEGF was almost undetectable in BVZ-treated groups after resection, but not in the non-resection group.
It was concluded that liver status was not impaired by BVZ even at the high-dose. Similarly, liver regeneration after extended hepatectomy in BVZ-treated animals was well-preserved. Extended liver resections may be encouraged in BVZ-treated patients due to its excellent tolerability and good liver regeneration status.
Abstract Background Cardiotrophin-1 (CT1) has been used to prevent cell death in different models of liver injury in rats. D-galactosamine induces cell death in culture rat and human hepatocytes. The ...present study evaluated the cytoprotective effects of CT1 in an experimental model of apoptosis induced by D-galactosamine in hepatocytes. Methods DNA fragmentation, calpain activity and Western blots of caspase-3, calpastatin and Stat3, and Akt phosphorylation were measured. Stat3 and Akt inhibitors were used to analyze the mechanisms of action of CT1. Results CT1 caused an increase in Stat3 and Akt phosphorylation and a decrease of DNA fragmentation, calpain activity, and caspase-3 induced by D-galactosamine. The reduction of calpain activity by CT1 was associated with an increase of calpastatin (its endogenous inhibitor). The effects of CT1 were also dependent on the activation of Sta3 or Akt. Conclusions CT1 decreases cell death through a mechanism related to Stat3 and Akt phosphorylation and activation of calpastatin in D-galactosamine-treated hepatocytes.
ABSTRACT
Acute kidney injury is a common complication of rhabdomyolysis. A better understanding of this syndrome may be useful to identify novel therapeutic targets because there is no specific ...treatment so far. Ferroptosis is an iron‐dependent form of regulated nonapoptotic cell death that is involved in renal injury. In this study, we investigated whether ferroptosis is associated with rhabdomyolysis‐mediated renal damage, and we studied the therapeutic effect of curcumin, a powerful antioxidant with renoprotective properties. Induction of rhabdomyolysis in mice increased serum creatinine levels, endothelial damage, inflammatory chemokines, and cytokine expression, alteration of redox balance (increased lipid peroxidation and decreased antioxidant defenses), and tubular cell death. Treatment with curcumin initiated before or after rhabdomyolysis induction ameliorated all these pathologic and molecular alterations. Although apoptosis or receptor‐interacting protein kinase (RIPK)3‐mediated necroptosis were activated in rhabdomyolysis, our results suggest a key role of ferroptosis. Thus, treatment with ferrostatin 1, a ferroptosis inhibitor, improved renal function in glycerol‐injected mice, whereas no beneficial effects were observed with the pan‐caspase inhibitor carbobenzoxy‐valyl‐alanyl‐aspartyl‐(O‐methyl)‐fluoromethylketone or in RIPK3‐deficient mice. In cultured renal tubular cells, myoglobin (Mb) induced ferroptosis‐sensitive cell death that was also inhibited by curcumin. Mechanistic in vitro studies showed that curcumin reduced Mb‐mediated inflammation and oxidative stress by inhibiting the TLR4/NF‐κB axis and activating the cytoprotective enzyme heme oxygenase 1. Our findings are the first to demonstrate the involvement of ferroptosis in rhabdomyolysis‐associated renal damage and its sensitivity to curcumin treatment. Therefore, curcumin may be a potential therapeutic approach for patients with this syndrome.—Guerrero‐Hue, M., García‐Caballero, C., Palomino‐Antolín, A., Rubio‐Navarro, A., Vázquez‐Carballo, C., Herencia, C., Martín‐Sanchez, D., Farré‐Alins, V., Egea, J., Cannata, P., Praga, M., Ortiz, A., Egido, J., Sanz, A. B., Moreno, J. A. Curcumin reduces renal damage associated with rhabdomyolysis by decreasing ferroptosis‐mediated cell death. FASEB J. 33,8961–8975 (2019). www.fasebj.org
Abstract
Background and Aims
Rhabdomyolysis is a pathological syndrome associated with massive and severe skeletal muscle injury. A common complication of rhabdomyolysis is acute kidney injury (AKI), ...a process that increases mortality of these patients. In rhabdomyolysis, myoglobin (Mb) is released from muscle cells and subsequently absorbed by the renal tubular epithelium causing a series of deleterious effects. TWEAK/Fn14 axis regulates renal inflammation and tubular cell death, pathogenic mechanisms involved in AKI. However, the role of this pathway in rhabdomyolysis-AKI has not been previously analyzed. In this study we analyzed the role of TWEAK/Fn14 axis in AKI associated with rhabdomyolysis and the AKI to chronic kidney disease (CKD) transition.
Method
We performed an experimental model of AKI associated to rhabdomyolysis by the intramuscular injection of 50% glycerol (10mg/Kg of weight) in Wild Type C57BL/6 (WT), TWEAK deficient (TWEAK -/-) and Fn14 deficient (Fn14 -/-) mice (male, 12 weeks old). In another set of experiments, C57BL/6 mice were intraperitoneally injected with anti-TWEAK mAb (10 mg/kg) or irrelevant isotype control IgG (10 mg/kg) every 48 hours. Blood and renal samples were taken 3 and 6 hours or 1, 3, 7 and 30 days after glycerol administration to perform gene expression studies by Real Time-PCR, immunohistochemistry analysis, protein expression studies by Western Blot and cell death assays (TUNEL). In vitro studies in murine tubular cells (MCTs) were also conducted to determine the effects of Mb on TWEAK/Fn14 expression, and the intracellular pathways involved in Mb-mediated regulation of TWEAK/Fn14 system.
Results
Fn14 renal expression was increased at earlier stages of rhabdomyolysis, correlating with decline of renal function. In MCTs cells, Mb induced Fn14 expression in a time- and dose-dependent manner, whereas TWEAK expression remained unchanged. The use of general antioxidants (N-Acetylcysteine) or Nrf2 inducers (curcumin or Sulforaphane) reduced Mb-mediated Fn14 upregulation in vivo and in cultured renal cells, whereas the opposite effect was observed with the HO-1 inhibitor tin protoporphyrin IX (SnPP). Furthermore, MAPK p38 and ERK1/2 inhibitors, such as SB203580 and PD98059 respectively, diminished Mb effects on Fn14 expression. Moreover, Mb enhanced pro-inflammatory effects of TWEAK in vitro. In the animal model, we observed that genetic TWEAK or Fn14 deficiency ameliorated rhabdomyolysis-related loss of renal function, reduced histological damage, tubular cell death, lipid peroxidation, and decreased the expression of inflammatory mediators (Ccl2, Tnf, Il6, Tlr4 and Ccl5), tubular injury markers (Havcr1 and Lcn2) and endothelial dysfunction (Edn1 and Icam1). TWEAK or Fn14 knockout mice also showed decreased long-term renal fibrosis (Collagen content and Fn1 expression) and inflammation (F4/80 macrophage infiltration and Ccl2 expression) 30 days after rhabdomyolysis induction. Treatment with a blocking anti-TWEAK monoclonal antibody reduced expression of Lcn2 and cell death at early stages of rhabdomyolysis and decreased long-term consequences, mainly fibrosis and inflammation.
Conclusion
Our data suggest that TWEAK/Fn14 axis is involved in AKI-rhabdomyolysis, as well as in the transition from AKI to CKD. TWEAK/Fn14 may be a potential therapeutic target to decrease harmful effects of rhabdomyolysis.
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