Thiazolidinediones (TZDs) are widely used to treat type 2 diabetes mellitus; however, their use is complicated by systemic fluid retention. Along the nephron, the pharmacological target of TZDs, peroxisome proliferator-activated receptor-gamma (PPARgamma, encoded by Pparg), is most abundant in the collecting duct. Here we show that mice treated with TZDs experience early weight gain from increased total body water. Weight gain was blocked by the collecting duct-specific diuretic amiloride and was also prevented by deletion of Pparg from the collecting duct, using Pparg (flox/flox) mice. Deletion of collecting duct Pparg decreased renal Na(+) avidity and increased plasma aldosterone. Treating cultured collecting ducts with TZDs increased amiloride-sensitive Na(+) absorption and Scnn1g mRNA (encoding the epithelial Na(+) channel ENaCgamma) expression through a PPARgamma-dependent pathway. These studies identify Scnn1g as a PPARgamma target gene in the collecting duct. Activation of this pathway mediates fluid retention associated with TZDs, and suggests amiloride might provide a specific therapy.

Co-cultures of endothelial cells (EC) and mesenchymal stem cells (MSC) in three-dimensional (3D) protein hydrogels can be used to recapitulate aspects of vasculogenesis in vitro. MSC provide paracrine signals that stimulate EC to form vessel-like structures, which mature as the MSC transition to the role of mural cells. In this study, vessel-like network formation was studied using 3D collagen/fibrin (COL/FIB) matrices seeded with embedded EC and MSC and cultured for 7 days. The EC:MSC ratio was varied from 5:1, 3:2, 1:1, 2:3 and 1:5. The matrix composition was varied at COL/FIB compositions of 100/0 (pure COL), 60/40, 50/50, 40/60 and 0/100 (pure FIB). Vasculogenesis was markedly decreased in the highest EC:MSC ratio, relative to the other cell ratios. Network formation increased with increasing fibrin content in composite materials, although the 40/60 COL/FIB and pure fibrin materials exhibited the same degree of vasculogenesis. EC and MSC were co-localized in vessel-like structures after 7 days and total cell number increased by approximately 70%. Mechanical property measurements showed an inverse correlation between matrix stiffness and network formation. The effect of matrix stiffness was further investigated using gels made with varying total protein content and by crosslinking the matrix using the dialdehyde glyoxal. This systematic series of studies demonstrates that matrix composition regulates vasculogenesis in 3D protein hydrogels, and further suggests that this effect may be caused by matrix mechanical properties. These findings have relevance to the study of neovessel formation and the development of strategies to promote vascularization in transplanted tissues.

.-The use of LiCl in clinical psychiatry is routinely complicated by overt nephrogenic diabetes insipidus (NDI), the mechanism of which is incompletely understood. In vitro studies indicate that lithium can induce renal medullary interstitial cell cyclooxygenase 2 (COX2) protein expression via inhibition of glycogen synthase kinase-3␤ (GSK-3␤). Both COX1 and COX2 are expressed in the kidney. Renal prostaglandins have been suggested to play an important role in lithium-induced polyuria. The present studies examined whether induction of the COX2 isoform contributes to LiCl-induced polyuria. Four days after initiation of lithium treatment in C57 BL/6J mice, urine volume increased in LiCl-treated mice by fourfold compared with controls (P Ͻ 0.0001) and was accompanied by decreased urine osmolality. This was temporally associated with increased renal COX2 protein expression and increased urinary PGE 2 excretion, whereas COX1 levels remained unchanged. COX2 inhibition significantly blunted lithium-induced polyuria (P Ͻ 0.0001) and reduced urinary PGE2 levels. Lithium-associated polyuria was also seen in COX1Ϫ/Ϫ mice and was associated with increased urinary PGE2. COX2 inhibition completely prevented polyuria and PGE2 excretion in COX1Ϫ/Ϫ mice, suggesting that COX2, but not COX1, plays a critical role in lithium-induced polyuria. Lithium also induced renal medullary COX2 protein expression in congenitally polyuric antidiuretic hormone (AHD)-deficient rats, demonstrating that lithiuminduced COX2 protein expression is not secondary to altered ADH levels or polyuria. Lithium also decreased renal medullary GSK-3␤ activity, and this was temporally related to increased COX2 expression in the kidney from lithium-treated mice, consistent with a tonic in vivo suppression of COX2 expression by GSK-3 activity. In conclusion, these findings temporally link decreased GSK-3 activity to enhanced renal COX2 expression and COX2-derived urine PGE2 excretion. Suppression of COX2-derived PGE2 blunts lithium-associated polyuria. prostaglandin E2; urine osmolality LITHIUM, A METALLIC MONOVALENT cation, has been used therapeutically for more than 150 years and remains an important pharmacotherapeutic agent for treatment of bipolar disorder and Alzheimer's disease (10,24,34). It is estimated that ϳ1 of every 1,000 individuals in the population is on lithium treatment (33). The use of lithium is frequently complicated by impaired renal water reabsorption, resulting in nephrogenic diabetes insipidus (NDI) (3). The mechanism by which lithium induces diabetes insipidus is incompletely understood. Sugawara et al. (31) reported that after LiCl treatment, rats exhibit increased urinary PGE 2 excretion. They also found the nonselective cyclooxygenase-inhibiting NSAID indomethacin reduced lithium-induced PGE 2 excretion and polyuria, suggesting that a diuretic cyclooxygenase product may be involved in lithium-associated diabetes insipidus. COX2, an inducible isoform of cyclooxygenase, is expressed in the kidney together with COX1, a constitutively exp...

Prostaglandin E2 (PGE 2 ), a major product of cyclooxygenase, exerts its functions by binding to four G protein-coupled receptors (EP1-4) and has been implicated in modulating angiogenesis. The present study examined the role of the EP4 receptor in regulating endothelial cell proliferation, migration, and tubulogenesis. Primary pulmonary microvascular endothelial cells were isolated from EP4 flox/flox mice and were rendered null for the EP4 receptor with adenoCre virus. Whereas treatment with PGE 2 or the EP4 selective agonists PGE 1 -OH and ONO-AE1-329 induced migration, tubulogenesis, ERK activation and cAMP production in control adenovirus-transduced endothelial EP4 flox/flox cells, no effects were seen in adenoCre-transduced EP4 flox/flox cells. The EP4 agonist-induced endothelial cell migration was inhibited by ERK, but not PKA inhibitors, defining a functional link between PGE 2 -induced endothelial cell migration and EP4-mediated ERK signaling. Finally, PGE 2 , as well as PGE 1 -OH and ONO-AE1-329, also promoted angiogenesis in an in vivo sponge assay providing evidence that the EP4 receptor mediates de novo vascularization in vivo.Angiogenesis, the process of new blood vessel formation from pre-existing vessels, is a multistep event that requires endothelial cell proliferation, migration, and tube formation. Angiogenesis is controlled by diverse factors, including cytokines, growth factors, as well as cyclooxygenase-2-derived eicosanoids (1, 2). The pro-angiogenic effects of cyclooxygenase-2 are mediated primarily by three products of arachidonic acid metabolism: thromboxane A 2 , prostaglandin E 2 (PGE 2 ), 2 and prostaglandin I 2 . These pro-angiogenic eicosanoids directly stimulate the synthesis of angiogenic factors, promote vascular sprouting, migration, tube formation, as well as enhance endothelial cell survival (1, 2).PGE 2 exerts its cellular effects by binding to four distinct E-prostanoid receptors (EP1-4) that belong to the family of seven transmembrane G protein-coupled rhodopsin-type receptors (3). Even though there is similar signaling mechanisms among these receptors, it is clear that each receptor has different and often opposing biological effects (4). For example, although the EP2 and EP4 receptors are both Gs coupled receptors and up-regulate intracellular cAMP levels, they mediate differential phosphorylation of cAMP response element-binding proteins (5). In addition, following activation, these two receptors exert different downstream effects on important intracellular mediators, including the PI3K and ERK pathways (6, 7). Moreover, the EP3 receptor usually counteracts EP2-and EP4-mediated up-regulation of cAMP by preferentially coupling to G i proteins (3).Some information regarding the role of PGE 2 in angiogenesis has been obtained using cancer models in mice where the receptors have been deleted by homologous recombination. In this context, mice lacking the EP2 receptor produce significantly fewer and less vascularized tumors than wild type mice in a two-stage skin carcinogenesi...

Phospholipid transfer protein (PLTP), also known as lipid transfer protein 2 (LTP-2), mediates a transfer of phospholipids between high-density lipoproteins (HDL). The molecular and macromolecular specificities of recombinant human PLTP were studied using a fluorometric assay based on the excimer fluorescence of pyrenyl lipids. To determine lipoprotein specificity of PLTP, donor very low density lipoproteins (VLDL), low-density lipoproteins (LDL), and HDL were labeled with 1-palmitoyl-2-[10-(1-pyrenyl)decanoyl]phosphatidylcholine (PPyDPC) and incubated with unlabeled acceptor VLDL, LDL, and HDL in every pairwise combination. The highest rate of PPyDPC transfer mediated by PLTP occurred between donor HDL and acceptor HDL. Reassembled HDL (rHDL) consisting of 1-palmitoyl-2-oleoylphosphatidylcholine, apolipoprotein A-I, and pyrene lipids (100:1:4) were used to demonstrate that PLTP transfers diacylglyceride > phosphatidic acid > sphingomyelin > phosphatidylcholine (PC) > phosphatidylglycerol > cerobroside > phosphatidylethanolamine. Thus, PLTP transfers a variety of lipids with two carbon chains and a polar head group. Unsaturation of one PC acyl chain greatly increased transfer rate, whereas increasing chain length and exchanging sn-1/sn-2 position had only small effects. The rate of PPyDPC transfer by PLTP decreases with increasing free cholesterol content in rHDL and with decreasing HDL size. In contrast to spontaneous transfer, PLTP mediates the accumulation of PC in small rHDL particles. PLTP may be important in vivo in the recycling of PC from mature HDL to nascent HDL, the latter of which are the initial acceptors of cholesterol from peripheral tissue for reverse cholesterol transport to the liver.

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The survival of renal medullary interstitial cells (RMICs) requires their adaptation to rapid shifts in ambient tonicity normally occurring in the renal medulla. Previous studies determined that cyclooxygenase-2 (COX 2) activation is critical for this adaptation. The present studies find that these adaptive mechanisms are dampened by the simultaneous activation of an apoptotic pathway linked to a glycogen synthase kinase 3␤ (GSK 3␤). Inhibition of GSK 3 by LiCl or specific small molecule GSK inhibitors increased RMIC survival following hypertonic stress, and transduction of RMICs with a constitutively active GSK 3␤ (AdGSK 3␤A9) significantly increased apoptosis, consistent with a proapoptotic role of GSK 3␤. Following GSK 3␤ inhibition, increased survival was accompanied by increased COX 2 expression and COX 2 reporter activity. In contrast, GSK 3␤ overexpression reduced COX 2 reporter activity. Importantly, enhanced RMIC survival produced by GSK 3␤ inhibition was completely dependent on COX 2 because it was abolished by a COX 2-specific inhibitor, SC58236. The signaling pathway by which GSK 3␤ suppresses COX 2 expression was then explored. GSK 3␤ inhibition increased both NFB and ␤-catenin activity associated with decreased IB and increased ␤-catenin levels. The increase in COX 2 following GSK 3␤ inhibition was entirely blocked by NFB inhibition using mutant IB adenovirus. However, adenoviral overexpression of ␤-catenin did not increase COX 2 levels. These findings suggest that GSK 3␤ negatively regulates COX 2 expression and that GSK 3␤ inhibitors protect RMICs from hypertonic stress via induction of NFB-COX 2-dependent pathway.To survive and function normally, renal medullary cells rely on a unique ability to withstand the rapidly shifting osmotic environment present in the renal medulla (2). Failure to adapt to the harsh environment in the renal medulla contributes to the development of papillary necrosis, observed following nonsteroidal anti-inflammatory drug use, pregnancy, and diabetes mellitus (3). Hypertonic stress activates two opposing cellular signaling cascades that either lead to cell death or promote cell survival. The balance between these two pathways determines cell fate. Several mechanisms have been proposed to contribute to the ability of renal medullary cells to survive hypertonic stress, including accumulation of organic osmolytes (4, 5) and induction of heat shock proteins (6, 7). In addition, recent studies demonstrate that renal medullary interstitial cells (RMICs) 1 depend on robust COX 2 activity to adapt to hypertonic stress, both in vitro and in vivo (2,8,9). Conversely, the mechanisms contributing to hypertonic stress-induced RMIC cell death are less clearly defined.GSK 3␤ signaling has been implicated in a variety of biological processes associated with altered cell survival and differentiation, including Wnt-associated developmental patterning and ␤-catenin-driven tumorigenesis (10, 11). Recent studies demonstrate that the GSK 3␤ inhibitor lithium protects neurons from stress-indu...

FEVAR and open repair have similar short-term outcomes but have diverging long-term outcomes that may be secondary to the selection bias of FEVAR being offered to high-risk patients. FEVAR is a favorable option in high-risk patients, and open repair remains viable as the gold standard.