Characterization of the renal function in Epac1 (RapGef3) knockout mice

The discovery of the exchange proteins directly activated by cAMP (Epac) has renewed our knowledge of intracellular cAMP signaling. The Epac1 isoform (RapGef3) shows high expression level in the kidney. In vitro studies have suggested multiple roles for Epac in the renal function. The development of an animal model were Epac1 is knocked out (Epac1-/- mice), however, allows for in vivo studies on Epac1 possible renal functions. In the present study Epac1-/- mice were compared to their reference wild type (WT) littermates, during baseline conditions as well as after per oral water load with and without presence of the antidiuretic vasopressin analog desmopressin. Under these conditions we measured renal excretion and/or clearance of creatinine, osmolytes, electrolytes, urea, cAMP and albumin. It turned out that Epac1-/- mice showed a normal ability to dilute their urine; Urine osmolality after water load combined with intra-peritoneal saline injection was 232 ± 14.90 in WT and 249 ± 7.377 in Epac1-/- mice. However, the effect of desmopressin on urine osmolality was significantly attenuated in Epac1-/- mice; Urine osmolality after water load and desmopressin was 598 ± 88.37 in WT and 443 ± 33.52 in Epac1-/- animals, with the increase (desmopressin relative to saline) being 163 % in WT and 77 % in Epac1-/- mice. The present study showed that a reduced response to desmopressin could not be explained by under-expression of either the AVP-stimulated urea transporter UT-A1 or the water channel AQP-2. A possibility is that Epac1 is required for optimal trafficking of AQP-2 to the apical membrane. Moreover, creatinine clearance, used as an estimate of glomerular filtration rate, was significantly increased in Epac1-/- mice following water loading (WT; 397 ± 44.98 µl/min vs. Epac1-/- 550 ± 19.52 µl/min). This difference in was not seen after treatment with furosemide (WT; 553 ± 121.9 µl/min vs. Epac1-/- 606 ± 64.01 µl/min), which blocks the Na+ -K+ -2Cl- co-transporter, and thus the tubulo-glomerular feedback response on vascular tone in the afferent arteriole. Urine and plasma analysis demonstrated no difference in the fractional clearance of osmolytes, creatinine, Na+ , K+ , and urea between WT and Epac1-/- animals. Epac1-/- mice did not demonstrate any sign of proteinuria, suggesting an intact glomerular filtration barrier. We conclude that renal functions are generally well preserved in Epac1-/- mice. These mice do, however, exhibit a moderate polydipsia and polyuria due to a perturbation of the effect of vasopressin on tubular water reabsorption. A role of Epac in the regulation of GFR at the level of macula densa is also suggested.Master i Medisinsk biologiMAMD-MEDBIBMED39

Cyanobacteria as a Source for Novel Anti-Leukemic Compounds

Cyanobacteria are an inspiring source of bioactive secondary metabolites. These bioactive agents are a diverse group of compounds which are varying in their bioactive targets, the mechanisms of action, and chemical structures. Cyanobacteria from various environments, especially marine benthic cyanobacteria, are found to be rich sources for the search for novel bioactive compounds. Several compounds with anticancer activities have been discovered from cyanobacteria and some of these have succeeded to enter the clinical trials. Varying anticancer agents are needed to overcome increasing challenges in cancer treatments. Different search methods are used to reveal anticancer compounds from natural products, but cell based methods are the most common. Cyanobacterial bioactive compounds as agents against acute myeloid leukemia are not well studied. Here we examined our new results combined with previous studies of anti-leukemic compounds from cyanobacteria with emphasis to reveal common features in strains producing such activity. We report that cyanobacteria harbor specific anti-leukemic compounds since several studied strains induced apoptosis against AML cells but were inactive against non-malignant cells like hepatocytes. We noted that particularly benthic strains from the Baltic Sea, such as Anabaena sp., were especially potential AML apoptosis inducers. Taken together, this review and re-analysis of data demonstrates the power of maintaining large culture collections for the search for novel bioactivities, and also how anti-AML activity in cyanobacteria can be revealed by relatively simple and low-cost assays.Peer reviewe

Characterization of the renal function in Epac1 (RapGef3) knockout mice

The discovery of the exchange proteins directly activated by cAMP (Epac) has renewed our knowledge of intracellular cAMP signaling. The Epac1 isoform (RapGef3) shows high expression level in the kidney. In vitro studies have suggested multiple roles for Epac in the renal function. The development of an animal model were Epac1 is knocked out (Epac1-/- mice), however, allows for in vivo studies on Epac1 possible renal functions. In the present study Epac1-/- mice were compared to their reference wild type (WT) littermates, during baseline conditions as well as after per oral water load with and without presence of the antidiuretic vasopressin analog desmopressin. Under these conditions we measured renal excretion and/or clearance of creatinine, osmolytes, electrolytes, urea, cAMP and albumin. It turned out that Epac1-/- mice showed a normal ability to dilute their urine; Urine osmolality after water load combined with intra-peritoneal saline injection was 232 ± 14.90 in WT and 249 ± 7.377 in Epac1-/- mice. However, the effect of desmopressin on urine osmolality was significantly attenuated in Epac1-/- mice; Urine osmolality after water load and desmopressin was 598 ± 88.37 in WT and 443 ± 33.52 in Epac1-/- animals, with the increase (desmopressin relative to saline) being 163 % in WT and 77 % in Epac1-/- mice. The present study showed that a reduced response to desmopressin could not be explained by under-expression of either the AVP-stimulated urea transporter UT-A1 or the water channel AQP-2. A possibility is that Epac1 is required for optimal trafficking of AQP-2 to the apical membrane. Moreover, creatinine clearance, used as an estimate of glomerular filtration rate, was significantly increased in Epac1-/- mice following water loading (WT; 397 ± 44.98 µl/min vs. Epac1-/- 550 ± 19.52 µl/min). This difference in was not seen after treatment with furosemide (WT; 553 ± 121.9 µl/min vs. Epac1-/- 606 ± 64.01 µl/min), which blocks the Na+ -K+ -2Cl- co-transporter, and thus the tubulo-glomerular feedback response on vascular tone in the afferent arteriole. Urine and plasma analysis demonstrated no difference in the fractional clearance of osmolytes, creatinine, Na+ , K+ , and urea between WT and Epac1-/- animals. Epac1-/- mice did not demonstrate any sign of proteinuria, suggesting an intact glomerular filtration barrier. We conclude that renal functions are generally well preserved in Epac1-/- mice. These mice do, however, exhibit a moderate polydipsia and polyuria due to a perturbation of the effect of vasopressin on tubular water reabsorption. A role of Epac in the regulation of GFR at the level of macula densa is also suggested

A liposomal formulation of simvastatin and doxorubicin for improved cardioprotective and anti-cancer effect

Anthracyclines such as doxorubicin (Dox) are the preferred chemotherapeutics for several cancers. However, Dox-induced cardiotoxicity limits its therapeutic potential. Liposomal encapsulation of Dox has been used for patients with risk to develop Dox induced cardiotoxicity but does not surpass the efficacy of the unencapsulated drug. Statins are widely used as cholesterol lowering drugs and have also demonstrated cardioprotective activity in cancer patients undergoing Dox therapy. We developed a liposome loaded with Dox and simvastatin (Sim) and investigated their effect on cardiomyocytes and zebrafish larvae. Furthermore, we investigated if the doses required for cardioprotection compromised the cytotoxicity of Dox in mammary and prostate cancer cells. Combination of Sim and Dox reduced ROS generation in cardiomyocytes, both given as free drugs, or co-encapsulated in liposomes. In contrast, Sim potentiated ROS-generation and cytotoxic activity of Dox towards cancer cells also when co-encapsulated in liposomes. In zebrafish larvae, Sim treatment reduced Dox-induced cardiac affection, and the liposomes did not induce any sign of Dox-induced cardiotoxicity. Our results show that liposomal co-encapsulation of Sim and Dox can be an efficient way of further reducing the risk of cardiotoxic events of liposomal Dox, while retaining, or even potentiating the anti-cancer effect of Dox.publishedVersio

Synthesis and activities of new indolopyrrolobenzodiazepine derivatives toward Acute Myeloid Leukemia cells.

International audienceThe synthesis of new indolopyrrolobenzodiazepine derivatives is described. Six compounds were selected for evaluation of cytotoxicity towards acute myeloid leukemia (AML) cells and normal fibroblasts. One compound (29) showed selective AML cell death induction. Its action was only partly overcome by knock-down of p53 or Bcl-2 overexpression, suggesting a strong activation of intrinsic apoptotic pathways

A Kinase Inhibitor with Anti-Pim Kinase Activity is a Potent and Selective Cytotoxic Agent Toward Acute Myeloid Leukemia

International audienc

Epac1 null mice have nephrogenic diabetes insipidus with deficient corticopapillary osmotic gradient and weaker collecting duct tight junctions

Aim The cAMP‐mediator Epac1 (RapGef3) has high renal expression. Preliminary observations revealed increased diuresis in Epac1−/− mice. We hypothesized that Epac1 could restrict diuresis by promoting transcellular collecting duct (CD) water and urea transport or by stabilizing CD paracellular junctions to reduce osmolyte loss from the renal papillary interstitium. Methods In Epac1−/− and Wt C57BL/6J mice, renal papillae, dissected from snap‐frozen kidneys, were assayed for the content of key osmolytes. Cell junctions were analysed by transmission electron microscopy. Urea transport integrity was evaluated by urea loading with 40% protein diet, endogenous vasopressin production was manipulated by intragastric water loading and moderate dehydration and vasopressin type 2 receptors were stimulated selectively by i.p.‐injected desmopressin (dDAVP). Glomerular filtration rate (GFR) was estimated as [14C]inulin clearance. The glomerular filtration barrier was evaluated by urinary albumin excretion and microvascular leakage by the renal content of time‐spaced intravenously injected 125I‐ and 131I‐labelled albumin. Results Epac1−/− mice had increased diuresis and increased free water clearance under antidiuretic conditions. They had shorter and less dense CD tight junction (TJs) and attenuated corticomedullary osmotic gradient. Epac1−/− mice had no increased protein diet‐induced urea‐dependent osmotic diuresis, and expressed Wt levels of aquaporin‐2 (AQP‐2) and urea transporter A1/3 (UT‐A1/3). Epac1−/− mice had no urinary albumin leakage and unaltered renal microvascular albumin extravasation. Their GFR was moderately increased, unless when treated with furosemide. Conclusion Our results conform to the hypothesis that Epac1‐dependent mechanisms protect against diabetes insipidus by maintaining renal papillary osmolarity and the integrity of CD TJs

Epac1 null mice have nephrogenic diabetes insipidus with deficient corticopapillary osmotic gradient and weaker collecting duct tight junctions

Aim The cAMP‐mediator Epac1 (RapGef3) has high renal expression. Preliminary observations revealed increased diuresis in Epac1−/− mice. We hypothesized that Epac1 could restrict diuresis by promoting transcellular collecting duct (CD) water and urea transport or by stabilizing CD paracellular junctions to reduce osmolyte loss from the renal papillary interstitium. Methods In Epac1−/− and Wt C57BL/6J mice, renal papillae, dissected from snap‐frozen kidneys, were assayed for the content of key osmolytes. Cell junctions were analysed by transmission electron microscopy. Urea transport integrity was evaluated by urea loading with 40% protein diet, endogenous vasopressin production was manipulated by intragastric water loading and moderate dehydration and vasopressin type 2 receptors were stimulated selectively by i.p.‐injected desmopressin (dDAVP). Glomerular filtration rate (GFR) was estimated as [14C]inulin clearance. The glomerular filtration barrier was evaluated by urinary albumin excretion and microvascular leakage by the renal content of time‐spaced intravenously injected 125I‐ and 131I‐labelled albumin. Results Epac1−/− mice had increased diuresis and increased free water clearance under antidiuretic conditions. They had shorter and less dense CD tight junction (TJs) and attenuated corticomedullary osmotic gradient. Epac1−/− mice had no increased protein diet‐induced urea‐dependent osmotic diuresis, and expressed Wt levels of aquaporin‐2 (AQP‐2) and urea transporter A1/3 (UT‐A1/3). Epac1−/− mice had no urinary albumin leakage and unaltered renal microvascular albumin extravasation. Their GFR was moderately increased, unless when treated with furosemide. Conclusion Our results conform to the hypothesis that Epac1‐dependent mechanisms protect against diabetes insipidus by maintaining renal papillary osmolarity and the integrity of CD TJs