Phorbol esters, the activators of protein kinase C (PKC), induce apoptosis in androgen-sensitive LNCaP prostate cancer cells. The role of individual PKC isozymes as mediators of this effect has not been thoroughly examined to date. To study the involvement of the novel isozyme PKC␦, we used a replication-deficient adenovirus (PKC␦AdV), which allowed for a tightly controlled expression of PKC␦ in LNCaP cells. A significant reduction in cell number was observed after infection of LNCaP cells with PKC␦AdV. Overexpression of PKC␦ markedly enhanced the apoptotic effect of phorbol 12-myristate 13-acetate in LNCaP cells. PKC␦-mediated apoptosis was substantially reduced by the pan-caspase inhibitor z-VAD and by Bcl-2 overexpression. Importantly, and contrary to other cell types, PKC␦-mediated apoptosis does not involve its proteolytic cleavage by caspase-3, suggesting that allosteric activation of PKC␦ is sufficient to trigger apoptosis in LNCaP cells. In addition, phorbol ester-induced apoptosis was blocked by a kinase-deficient mutant of PKC␦, supporting the concept that PKC␦ plays an important role in the regulation of apoptotic cell death in LNCaP prostate cancer cells.

Acute tubular necrosis (ATN) caused by ischemia/reperfusion (I/R) during renal transplantation delays allograft function. Identification of factors that mediate protection and/or epithelium recovery could help to improve graft outcome. We studied the expression, regulation and role of hypoxia inducible factor 1-alpha (HIF-1 α), using in vitro and in vivo experimental models of I/R as well as human post-transplant renal biopsies. We found that HIF-1 α is stabilized in proximal tubule cells during ischemia and unexpectedly in late reperfusion, when oxygen tension is normal. Both inductions lead to gene expression in vitro and in vivo. In vitro interference of HIF-1 α promoted cell death and in vivo interference exacerbated tissue damage and renal dysfunction. In pos-transplant human biopsies, HIF-1 α was expressed only in proximal tubules which exhibited normal renal structure with a significant negative correlation with ATN grade. In summary, using experimental models and human biopsies, we identified a novel HIF-1 α induction during reperfusion with a potential critical role in renal transplant.

Pulse treatment of U-937 promonocytic cells with cadmium chloride (2 h at 200 M) provoked apoptosis and induced a rapid phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK ) as well as a late phosphorylation of extracellular signal-regulated protein kinases (ERK1/2). However, although the p38 MAPK -specific inhibitor SB203580 attenuated apoptosis, the process was not affected by the ERK-specific inhibitor PD98059. The attenuation of the cadmium-provoked apoptosis by SB203580 was a highly specific effect. In fact, the kinase inhibitor did not prevent the generation of apoptosis by heat shock and camptothecin, nor the generation of necrosis by cadmium treatment of glutathione-depleted cells, nor the cadmium-provoked activation of the stress response. The generation of apoptosis was preceded by intracellular H 2 O 2 accumulation and was accompanied by the disruption of mitochondrial transmembrane potential, both of which were inhibited by SB203580. On the other hand, the antioxidant agent butylated hydroxyanisole-inhibited apoptosis but did not prevent p38 MAPK phosphorylation. In a similar manner, p38 MAPK phosphorylation was not affected by the caspase inhibitors Z-VAD and DEVD-CHO, which nevertheless prevented apoptosis. These results indicate that p38 MAPK activation is an early and specific regulatory event for the cadmium-provoked apoptosis in promonocytic cells.Cadmium and other heavy metals are frequent environmental contaminants with well known mutagenic, carcinogenic, and teratogenic effects (1). Another property of heavy metals (and of other physical and chemical agents, such as heat and inhibitors of energy metabolism) is the capacity to induce the stress response, characterized by the synthesis and accumulation of heat-shock proteins (HSPs)

In the last decade, Acute Kidney Injury (AKI) diagnosis and therapy have not notably improved probably due to delay in the diagnosis, among other issues. Precocity and accuracy should be critical parameters in novel AKI biomarker discovery. microRNAs are key regulators of cell responses to many stimuli and they can be secreted to the extracellular environment. Therefore, they can be detected in body fluids and are emerging as novel disease biomarkers. We aimed to identify and validate serum miRNAs useful for AKI diagnosis and management. Using qRT-PCR arrays in serum samples, we determined miRNAs differentially expressed between AKI patients and healthy controls. Statistical and target prediction analysis allowed us to identify a panel of 10 serum miRNAs. This set was further validated, by qRT-PCR, in two independent cohorts of patients with relevant morbi-mortality related to AKI: Intensive Care Units (ICU) and Cardiac Surgery (CS). Statistical correlations with patient clinical parameter were performed. Our results demonstrated that the 10 selected miRNAs (miR-101-3p, miR-127-3p, miR-210-3p, miR-126-3p, miR-26b-5p, miR-29a-3p, miR-146a-5p, miR-27a-3p, miR-93-3p and miR-10a-5p) were diagnostic biomarkers of AKI in ICU patients, exhibiting areas under the curve close to 1 in ROC analysis. Outstandingly, serum miRNAs estimated before CS predicted AKI development later on, thus becoming biomarkers to predict AKI predisposition. Moreover, after surgery, the expression of the miRNAs was modulated days before serum creatinine increased, demonstrating early diagnostic value. In summary, we have identified a set of serum miRNAs as AKI biomarkers useful in clinical practice, since they demonstrate early detection and high diagnostic value and they recognize patients at risk.

Uncontrolled extracellular matrix (ECM) production by fibroblasts in response to injury contributes to fibrotic diseases, including idiopathic pulmonary fibrosis (IPF). Reactive oxygen species (ROS) generation is involved in the pathogenesis of IPF. Transforming growth factor-b1 (TGF-b1) stimulates the production of NADPH oxidase 4 (NOX4)-dependent ROS, promoting lung fibrosis (LF). Dysregulation of microRNAs (miRNAs) has been shown to contribute to LF. To identify miRNAs involved in redox regulation relevant for IPF, we performed arrays in human lung fibroblasts exposed to ROS. miR-9-5p was selected as the best candidate and we demonstrate its inhibitory effect on TGF-b receptor type II (TGFBR2) and NOX4 expression. Increased expression of miR-9-5p abrogates TGF-b1-dependent myofibroblast phenotypic transformation. In the mouse model of bleomycin-induced LF, miR-9-5p dramatically reduces fibrogenesis and inhibition of miR-9-5p and prevents its antifibrotic effect both in vitro and in vivo. In lung specimens from patients with IPF, high levels of miR-9-5p are found. In omentumderived mesothelial cells (MCs) from patients subjected to peritoneal dialysis (PD), miR-9-5p also inhibits mesothelial to myofibroblast transformation. We propose that TGF-b1 induces miR-9-5p expression as a self-limiting homeostatic response.