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

Análisis de los valores de hipertensión arterial en pacientes que emplean sistemas personalizados de dosificación.

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Intracellular prostaglandin E2 contributes to hypoxia-induced proximal tubular cell death

Proximal tubular cells (PTC) are particularly vulnerable to hypoxia-induced apoptosis, a relevant factor for kidney disease. We hypothesized here that PTC death under hypoxia is mediated by cyclooxygenase (COX-2)-dependent production of prostaglandin E2 (PGE2), which was confirmed in human proximal tubular HK-2 cells because hypoxia (1% O2)- induced apoptosis (i) was prevented by a COX-2 inhibitor and by antagonists of prostaglandin (EP) receptors and (ii) was associated to an increase in intracellular PGE2 (iPGE2) due to hypoxia-inducible factor-1?-dependent transcriptional up-regulation of COX-2. Apoptosis was also prevented by inhibitors of the prostaglandin uptake transporter PGT, which indicated that iPGE2 contributes to hypoxia-induced apoptosis (on the contrary, hypoxia/reoxygenation-induced PTC death was exclusively due to extracellular PGE2). Thus, iPGE2 is a new actor in the pathogenesis of hypoxia-induced tubular injury and PGT might be a new therapeutic target for the prevention of hypoxia-dependent lesions in renal diseases.Ministerio de Ciencia e InnovaciónUniversidad de AlcaláComunidad de Madri

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

Time-series proteomic study of the response of HK-2 cells to hyperglycemic, hypoxic diabetic-like milieu

During diabetes, renal proximal tubular cells (PTC) are exposed to a combination of high glucose and hypoxic conditions, which plays a relevant role in the development of diabetic kidney disease (DKD). In this work, a time-series proteomic study was performed to analyse the effect of a diabetic-like microenvironment induced changes on HK-2 cells, a human cell line derived from normal proximal tubular epithelial cells. Cells simultaneously exposed to high glucose (25 mM) and hypoxia (1% O-2) were compared to cells in control conditions for up to 48 h. Diabetic conditions increased the percentage of death cells after 24 and 48 h, but no differences in the protein/cell ratio were found. The relative protein quantification using dimethyl-labeling and UHPLC-MS/MS analysis allowed the identification of 317, 296 and 259 proteins at 5, 24 and 48 h, respectively. The combination of statistical and time expression profile analyses indicated an increased expression of proteins involved in glycolysis, and a decrease of cytoskeletal-related proteins. The exposure of HK-2 cells to high glucose and hypoxia reproduces some of the effects of diabetes on PTC and, with the limitations inherent toin vitrostudies, propose new mechanisms and targets to be considered in the management of DKD

Regulación de HIF-1α por microparticulas de células tubulares proximales renales. Papel de COX y PGE2

Las micro partículas (MPs) presentes en diferentes líquidos corporales, proceden de invaginaciones de la membrana celular. Engloban diferentes factores reguladores celulares y se las considera como un nuevo mecanismo de comunicación celular. Estudios de nuestro laboratorio, han demostrado el efecto regulatorio del factor inducible por hipoxia–1alpha (HIF- 1α un factor de transcripción génica) en células renales humanas de túbulo proximal (HK-2), tras el tratamiento con MPs procedentes de células endoteliales. Dicho aumento lleva asociado un mecanismo de amplificación de la señal mediado por la generación de nuevas MPs producidas por células HK-2, que regulan HIF-1α de manera independiente. El presente trabajo, demuestra que las células HK-2 producen MPs en condiciones basales y que actúan como un sistema paracrino de regulación de HIF-1α mediante una vía de señalización probablemente mediada por COX y PGE2. Teniendo en cuenta la importancia de la PGE2 y de HIF-1α en diferentes procesos de daño renal, consideramos que las MPs son un mecanismo novedoso de regulación de HIF-1α que debe ser estudiado con mayor profundidad en el contexto de la fisiología y patología rena

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