Intracellular prostaglandin E2 mediates cisplatin-induced proximal tubular cell death

Nephrotoxicity, particularly in the proximal tubule, limits the therapeutic efficacy of the antineoplastic drug cisplatin. The signaling mechanisms appear to be multifactorial, involving inflammation, oxidative stress, and caspase. Here we studied the role of intracellular prostaglandin E2 (iPGE2) in cisplatin's cytotoxicity in human proximal tubular HK-2 cells. Cisplatin-induced apoptotic cell death was prevented by inhibitors of the prostaglandin transporter (PGT) or by PGT knock-down or by pharmacologic inhibition of PGE2 EP receptors or cyclo-oxygenase-2 (COX-2). iPGE2 also increased in cisplatin-treated cells, which was probably due to increased expression of COX-2, microsomal PGE2 synthase-1 and PGT, and was prevented by inhibitors of PGT or COX-2. Thus iPGE2, most likely acting through intracellular EP receptors, mediates cisplatin-induced HK-2 cell death. Importantly, the tumoricidal effect of cisplatin on human cervical adenocarcinoma HeLa cells was not affected by a pharmacologic inhibitor of PGT. In conclusion, iPGE2 may play a significant role in the pathogenesis of cisplatin's nephrotoxicity and treatment with PGT inhibitors might represent a novel strategy in its preventionMinisterio de Ciencia e InnovaciónComunidad de Madri

PROSTAGLANDIN E2 stimulates cancer-related phenotypes in prostate cancer PC3 cells through cyclooxygenase-2

Cyclooxygenase (COX)?derived prostaglandin E2 (PGE2) affects many mechanisms that have been shown to play roles in carcinogenesis. Recently, we found that, in androgenindependent prostate cancer PC3 cells, PGE2 acts through an intracrine mechanism by which its uptake by the prostaglandin transporter (PGT) results in increased intracellular PGE2 (iPGE2), leading to enhanced cell proliferation, migration, invasion, angiogenesis, and loss of cell adhesion to collagen I. These iPGE2?mediated effects were dependent on hypoxia?inducible factor 1?? (HIF?1?), whose expression increased upon epidermal growth factor receptor (EGFR) transactivation by a subset of intracellular PGE2 receptors. Here, we aimed to study the role of COX in PGE2 protumoral effects in PC3 cells and found that the effects were prevented by inhibition of COX?2, which highlights its crucial role amplifying the levels of iPGE2. Treatment with exogenous PGE2 determined a transcriptional increase in COX?2 expression, which was abolished by genetic or pharmacologic inhibition of PGT. PGE2?induced increase in COX?2 expression and, thereby, in transcriptional increase in HIF?1? expression was due to EGFR activation, leading to the activation of Phosphoinositide 3-kinase/Akt, Extracellular signal -regulated kinases 1/2, p38 and Mitogen- and stress-activated protein kinase-1 (PI3K/Akt, Erk1/2, p38 and MSK?1). Collectively, the data suggest that EGFR?dependent COX?2 upregulation by a novel positive feedback loop triggered by iPGE2 underlies the intracrine pro?tumoral effects of PGE2 in PC3 cells. Therefore, this feedback loop may be relevant in prostate cancer for the maintenance of PGE2?dependent cancer cell growth through amplifying the activity of the COX?2 pathway.Ministerio de Ciencia e Innovació

Retinoic acid receptor-beta prevents cisplatin-induced proximal tubular cell death

Cisplatin toxicity in renal tubular epithelial cells limits the therapeutic efficacy of this antineoplastic drug. In cultured human proximal tubular HK-2 cells (PTC) a prostaglandin uptake transporter (PGT)-dependent increase in intracellular prostaglandin E2 (iPGE2) mediates cisplatin's toxicity (i.e. increased cell death and loss of cell proliferation) so that it is prevented by PGT inhibitors. Here we found in cisplatin-treated PTC that 4,4?-diisothiocyanostilbene-2,2?-disulfonic acid (DIDS), a PGT inhibitor, prevented cisplatin's toxicity but not the increase in iPGE2. Because expression of retinoic acid receptor-? (RAR-?) is dependent on iPGE2 and because RAR-? is a regulator of cell survival and proliferation, we hypothesized that RAR-? might mediate the protective effect of DIDS against cisplatin's toxicity in PTC. Our results confirmed this hypothesis because: i) protection of PTC by DIDS was abolished by RAR-? antagonist LE-135; ii) DIDS increased the expression of RAR-? in PTC and prevented its decrease in cisplatin-treated PTC but not in cisplatin-treated human cervical adenocarcinoma HeLa cells in which DIDS failed to prevent cisplatin's toxicity; iii) while RAR-? expression decreased in cisplatin-treated PTC, RAR-? over-expression prevented cisplatin's toxicity. RAR-? agonist CH55 or RAR pan-agonist all-trans retinoic acid did not prevent cisplatin's toxicity, which suggests that RAR-? does not protect PTC through activation of gene transcription. In conclusion, RAR-? might be a new player in cisplatin-induced proximal tubular injury and the preservation of its expression in proximal tubules through treatment with DIDS might represent a novel strategy in the prevention of cisplatin nephrotoxicity without compromising cisplatin's chemotherapeutic effect on cancer cells

Prostaglandin transporter PGT as a new pharmacological target in the prevention of inflammatory cytokine-induced injury in renal proximal tubular HK-2 cells

Aims: Inflammatory cytokines contribute to proximal tubular cell (PTC) injury leading to the deterioration of renal function and acute kidney injury (AKI) development. They also stimulate cyclo‑oxygenase-2 (COX-2)- dependent production and release to the extracellular medium of prostaglandin E2 (PGE2), a mediator of PTC injury. However, in several settings PGE2 re-uptake by prostaglandin transporter (PGT) is critical for PGE2- mediated PTC injury. Here we investigated several deleterious effects of pro-inflammatory cytokines in PTC and their prevention by PGT targeting. Main methods: In human kidney-2 (HK-2) PTC exposed to an inflammatory cytokine cocktail, consisting of interleukins (IL) IL-1α, IL-1β and IL-2, tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), were determined the changes in several parameters related to PTC injury, their dependency on PGE2 (through modulation by antagonists of PGE2 receptors) and the preventive effect of PGT inhibitor bromosulfophthalein. Key findings: The cytokine cocktail induced a COX-2-dependent increase in intracellular PGE2 (iPGE2) and cell death, together to a decrease in cell number and cell proliferation. There was also loss of adherent cells to collagen IV, changes in actin cytoskeleton and loss of monolayer integrity, together to an increase in paracellular permeability. All the changes were sensitive to antagonist of PGE2 receptors AH6809 and were fully prevented by bromosulfophthalein. Significance: These results indicate that PGT-, iPGE2-dependent mechanisms mediate inflammatory cytokineinduced HK-2 cell injury and suggest that treatment with PGT inhibitors might help to prevent AKI induced by sepsis, renal ischemia/reperfusion and other pathological conditions in which inflammatory cytokines contribute to PTC damageThis work was supported by a grant COVID-19 2021 2020/00003/ 016/001/009 from the Universidad de Alcala and a grant Ayudas a la Investigacion ´ Departamento de Biología UAM. This research is part of the project on COVID-19 and diabetes (REACT UE-CM2021-02) funded by the Community of Madrid in agreement with the University of Alcala, ´ and co-funded with REACT-EU resources from the European Regional Development Fund “A way to make Europe

Apoptosis and cell proliferation in proximal tubular cells exposed to apoptotic bodies. Novel pathophysiological implications in cisplatin-induced renal injury

The therapeutic efficacy of the antineoplastic drug cisplatin is limited by its nephrotoxicity, which affects particularly to proximal tubular cells (PTC). Cisplatin-induced cytotoxicity appears to be multifactorial and involves inflammation, oxidative stress as well as apoptosis. We have recently shown that the cyclo-oxygenase-2 (COX2)/intracellular prostaglandin E2 (iPGE2)/EP receptor pathway mediates the apoptotic effect of cisplatin on human proximal tubular HK-2 cells. Here, we studied the effects on HK-2 cells of apoptotic bodies (ABs) generated after treatment of HK-2 cells with cisplatin. We found that ABs inhibited cell growth, induced apoptosis and increased COX-2 expression and iPGE2 in ABs-recipient HK-2 cells. Inhibition of the COX-2/iPGE2/EP receptor pathway in these cells prevented the effects of ABs without interfering with their internalization. Interestingly, 2nd generation ABs (i.e. ABs released by cells undergoing apoptosis upon treatment with ABs) did not trigger apoptosis in naïve HK-2 cells, and stimulated cell proliferation through the COX-2/iPGE2/EP receptor pathway. These results suggest that ABs, through iPGE2-dependent mechanisms, might have a relevant role in the natural history of cisplatin-induced acute kidney failure because they contribute first to the propagation of the noxious effects of cisplatin to non-injured PTC and then to the promotion of the proliferative tubular response required for proximal tubule repair. Since iPGE2 also mediates both cisplatin-induced HK-2 cell apoptosis, intervention in the COX-2/iPGE2/EP receptor pathway might provide us with new therapeutic avenues in patients with cisplatin-induced acute kidney injuryThis work was supported in part from grants from the Spanish Ministerio de Ciencia e Innovación, Spain (SAF2011-26838 and SAF2014-53218-R), Instituto de Salud Carlos III, Spain (PI15/02139) -Fondo Europeo de Desarrollo Regional, European-Union (FEDER)- and Comunidad Autónoma de Madrid, Spain -Programa de Actividades I+D en Biomedicina 2017- (B2017-BMD-3686). Coral Garcia-Pastor is recipient of a FPU fellowship from the University of Alcal

Mechanism and Consequences of The Impaired Hif-1α Response to Hypoxia in Human Proximal Tubular HK-2 Cells Exposed to High Glucose

Renal hypoxia and loss of proximal tubular cells (PTC) are relevant in diabetic nephropathy. Hypoxia inhibits hypoxia-inducible factor-1α (HIF-1α) degradation, which leads to cellular adaptive responses through HIF-1-dependent activation of gene hypoxia-responsive elements (HRE). However, the diabetic microenvironment represses the HIF-1/HRE response in PTC. Here we studied the mechanism and consequences of impaired HIF-1α regulation in human proximal tubular HK-2 cells incubated in hyperglycemia. Inhibition at diferent levels of the canonical pathway of HIF-1α degradation did not activate the HIF-1/HRE response under hyperglycemia, except when proteasome was inhibited. Further studies suggested that hyperglycemia disrupts the interaction of HIF-1α with Hsp90, a known cause of proteasomal degradation of HIF-1α. Impaired HIF-1α regulation in cells exposed to hyperglycemic, hypoxic diabetic-like milieu led to diminished production of vascular endothelial growth factor-A and inhibition of cell migration (responses respectively involved in tubular protection and repair). These efects, as well as impaired HIF-1α regulation, were reproduced in normoglycemia in HK-2 cells incubated with microparticles released by HK-2 cells exposed to diabetic-like milieu. In summary, these results highlight the role of proteasome-dependent mechanisms of HIF-1α degradation on diabetesinduced HK-2 cells dysfunction and suggest that cell-derived microparticles may mediate negative efects of the diabetic milieu on PTCThis work was supported by grants SAF2014-53218-R from the Spanish Ministerio de Ciencia e Innovación and RTI2018-095872-B-C21/ERDF by AEI. Coral Garcia-Pastor is the recipient of a FPU fellowship from the University of Alcala

Apoptosis and cell proliferation in proximal tubular cells exposed to apoptotic bodies. Novel pathophysiological implications in cisplatin-induced renal injury

The therapeutic efficacy of the antineoplastic drug cisplatin is limited by its nephrotoxicity, which affects particularly to proximal tubular cells (PTC). Cisplatin-induced cytotoxicity appears to be multifactorial and involves inflammation, oxidative stress as well as apoptosis. We have recently shown that the cyclo-oxygenase-2 (COX-2)/intracellular prostaglandin E-2 (iPGE(2))/EP receptor pathway mediates the apoptotic effect of cisplatin on human proximal tubular HK-2 cells. Here, we studied the effects on HK-2 cells of apoptotic bodies (ABs) generated after treatment of HK-2 cells with cisplatin. We found that ABs inhibited cell growth, induced apoptosis and increased COX-2 expression and iPGE(2) in ABs-recipient HK-2 cells. Inhibition of the COX-2/iPGE(2)/EP receptor pathway in these cells prevented the effects of ABs without interfering with their internalization. Interestingly, 2nd generation ABs (i.e. ABs released by cells undergoing apoptosis upon treatment with ABs) did not trigger apoptosis in naive HK-2 cells, and stimulated cell proliferation through the COX-2/iPGE(2)/EP receptor pathway. These results suggest that ABs, through iPGE(2)-dependent mechanisms, might have a relevant role in the natural history of cisplatin-induced acute kidney failure because they contribute first to the propagation of the noxious effects of cisplatin to non-injured PTC and then to the promotion of the proliferative tubular response required for proximal tubule repair. Since iPGE(2) also mediates both cisplatin-induced HK-2 cell apoptosis, intervention in the COX-2/iPGE(2)/EP receptor pathway might provide us with new therapeutic avenues in patients with cisplatin-induced acute kidney injury.Ministerio de Ciencia e InnovaciónInstituto de Salud Carlos IIIComunidad Autónoma de Madri

Mechanism and Consequences of The Impaired Hif-1alfa Response to Hypoxia in Human Proximal Tubular HK-2 Cells Exposed to High Glucose

Renal hypoxia and loss of proximal tubular cells (PTC ) are relevant in diabetic nephropathy. Hypoxia inhibits hypoxia-inducible factor-1? (HIF-1?) degradation, which leads to cellular adaptive responses through HIF-1-dependent activation of gene hypoxia-responsive elements (HRE). However, the diabetic microenvironment represses the HIF-1/HRE response in PTC. Here we studied the mechanism and consequences of impaired HIF-1? regulation in human proximal tubular HK-2 cells incubated in hyperglycemia. Inhibition at different levels of the canonical pathway of HIF-1? degradation did not activate the HIF-1/HRE response under hyperglycemia, except when proteasome was inhibited. Further studies suggested that hyperglycemia disrupts the interaction of HIF-1? with Hsp90, a known cause of proteasomal degradation of HIF-1?. Impaired HIF-1? regulation in cells exposed to hyperglycemic, hypoxic diabetic-like milieu led to diminished production of vascular endothelial growth factor-A and inhibition of cell migration (responses respectively involved in tubular protection and repair). These effects, as well as impaired HIF-1? regulation, were reproduced in normoglycemia in HK-2 cells incubated with microparticles released by HK-2 cells exposed to diabetic-like milieu. In summary, these results highlight the role of proteasome-dependent mechanisms of HIF-1? degradation on diabetes-induced HK-2 cells dysfunction and suggest that cell-derived microparticles may mediate negative effects of the diabetic milieu on PTC.Ministerio de Ciencia e Innovació

Exploratory Metabolomic Analysis Based on Reversed-Phase Liquid Chromatography-Mass Spectrometry to Study an In Vitro Model of Hypoxia-Induced Metabolic Alterations in HK-2 Cells

Oxygen deficiency in cells, tissues, and organs can not only prevent the proper development of biological functions but it can also lead to several diseases and disorders. In this sense, the kidney deserves special attention since hypoxia can be considered an important factor in the pathophysiology of both acute kidney injury and chronic kidney disease. To provide better knowledge to unveil the molecular mechanisms involved, new studies are necessary. In this sense, this work aims to study, for the first time, an in vitro model of hypoxia-induced metabolic alterations in human proximal tubular HK-2 cells because renal proximal tubules are particularly susceptible to hypoxia. Different groups of cells, cultivated under control and hypoxia conditions at 0.5, 5, 24, and 48 h, were investigated using untargeted metabolomic approaches based on reversed-phase liquid chromatography-mass spectrometry. Both intracellular and extracellular fluids were studied to obtain a large metabolite coverage. On the other hand, multivariate and univariate analyses were carried out to find the differences among the cell groups and to select the most relevant variables. The molecular features identified as affected metabolites were mainly amino acids and Amadori compounds. Insights about their biological relevance are also provided

Aligned copper nanowires as a cut-and-paste exclusive electrochemical transducer for free-enzyme highly selective quantification of intracellular hydrogen peroxide in cisplatin-treated cells

The role and reliable quantification of intracellular hydrogen peroxide during cancer therapy constitutes an unexplored and fascinating application. In this work, we report the fabrication of vertically aligned copper nanowires (v-CuNWs) using electrosynthesis on templates, and their application as a cut-and-paste exclusive and flexible electrochemical transducer. This easily adaptable electrodic platform is demonstrated for a fast, simple and free-enzyme selective quantification of intracellular hydrogen peroxide in Cisplatin-treated human renal HK-2 cells. The v-CuNWs sensor was compared with an HRP-enzyme-based biosensor showing excellent correlation and indicates the good selectivity and analytical performance of the v-CuNWs. This sensing approach opens novel avenues for monitoring cell death processes and shows the potential of H2O2 as a cellular damage biomarker, with a clear potency for further developments for in vitro diagnosis and its implication in cancer therapy.Comunidad de MadridMinisterio de Economía y CompetitividadMinisterio de Ciencia e Innovació