Urinary exosomes reveal protein signatures in hypertensive patients with albuminuria

Albuminuria is an indicator of cardiovascular risk and renal damage in hypertensive individuals. Chronic renin-angiotensin system (RAS) suppression facilitates blood pressure control and prevents development of new-onset-albuminuria. A significant number of patients, however, develop albuminuria despite chronic RAS blockade, and the physiopathological mechanisms are underexplored. Urinary exosomes reflect pathological changes taking place in the kidney. The objective of this work was to examine exosomal protein alterations in hypertensive patients with albuminuria in the presence of chronic RAS suppression, to find novel clues underlying its development. Patients were followed-up for three years and were classified as: a) patients with persistent normoalbuminuria; b) patients developing de novo albuminuria; and c) patients with maintained albuminuria. Exosomal protein alterations between groups were identified by isobaric tag quantitation (iTRAQ). Confirmation was approached by target analysis (SRM). In total, 487 proteins were identified with high confidence. Specifically, 48 proteins showed an altered pattern in response to hypertension and/or albuminuria. Out of them, 21 proteins interact together in three main functional clusters: glycosaminoglycan degradation, coagulation and complement system, and oxidative stress. The identified proteins constitute potential targets for drug development and may help to define therapeutic strategies to evade albuminuria progression in hypertensive patients chronically treated.Instituto de Salud Carlos III, fondos FEDER/FSE (PI11/01401, PI13/01873, PI14/01841, IF08/3667-1, PI11-02239, PI 14/0917, PI11/02432, PI13/01746, PI14/01650, PI16/01334, PT13/0001/0013, CP09/00229, CP15/00129, CPII15/00027), Fundacion SENEFRO, Fundacion Conchita Rabago de Jimenez Diaz, and Redes Tematicas de Investigacion Cooperativa (fondos FEDER/FSE, RD12/0021/0001, RD12/0042/0071). These results are lined up with the Spanish initiative on the Human Proteome Project (SpHPP).S

Plasma Molecular Signatures in Hypertensive Patients With Renin-Angiotensin System Suppression: New Predictors of Renal Damage and De Novo Albuminuria Indicators

Albuminuria is a risk factor strongly associated with cardiovascular disease, the first cause of death in the general population. It is well established that renin-angiotensin system suppressors prevent the development of new-onset albuminuria in naïf hypertensive patients and diminish its excretion, but we cannot forget the percentage of hypertensive patients who develop de novo albuminuria. Here, we applied multiple proteomic strategy with the purpose to elucidate specific molecular pathways involved in the pathogenesis and provide predictors and chronic organ damage indicators. Briefly, 1143 patients were followed up for a minimum period of 3 years. One hundred and twenty-nine hypertensive patients chronically renin-angiotensin system suppressed were recruited, classified in 3 different groups depending on their albuminuria levels (normoalbuminuria, de novo albuminuria, and sustained albuminuria), and investigated by multiple proteomic strategies. Our strategy allowed us to perform one of the deepest plasma proteomic analysis to date, which has shown 2 proteomic signatures: (1) with predictive value of de novo albuminuria and (2) sustained albuminuria indicator proteins. These proteins are involved in inflammation, immune as well as in the proteasome activation occurring in situations of endoplasmic reticulum stress. Furthermore, these results open the possibility of a future strategy based on anti-immune therapy to treat hypertension which could help to prevent the development of albuminuria and, hence, the progression of kidney damage.N

Differential Role of Human Choline Kinase α and β Enzymes in Lipid Metabolism: Implications in Cancer Onset and Treatment

11 pages, 6 figures, 1 table.Background The Kennedy pathway generates phosphocoline and phosphoethanolamine through its two branches. Choline Kinase (ChoK) is the first enzyme of the Kennedy branch of synthesis of 1phosphocholine, the major component of the plasma membrane. ChoK family of proteins is composed by ChoKα and ChoKβ isoforms, the first one with two different variants of splicing. Recently ChoKα has been implicated in the carcinogenic process, since it is over-expressed in a variety of human cancers. However, no evidence for a role of ChoKβ in carcinogenesis has been reported. Methodology/Principal Findings Here we compare the in vitro and in vivo properties of ChoKα1 and ChoKβ in lipid metabolism, and their potential role in carcinogenesis. Both ChoKα1 and ChoKβ showed choline and ethanolamine kinase activities when assayed in cell extracts, though with different affinity for their substrates. However, they behave differentially when overexpressed in whole cells. Whereas ChoKβ display an ethanolamine kinase role, ChoKα1 present a dual choline/ethanolamine kinase role, suggesting the involvement of each ChoK isoform in distinct biochemical pathways under in vivo conditions. In addition, while overexpression of ChoKα1 is oncogenic when overexpressed in HEK293T or MDCK cells, ChoKβ overexpression is not sufficient to induce in vitro cell transformation nor in vivo tumor growth. Furthermore, a significant upregulation of ChoKα1 mRNA levels in a panel of breast and lung cancer cell lines was found, but no changes in ChoKβ mRNA levels were observed. Finally, MN58b, a previously described potent inhibitor of ChoK with in vivo antitumoral activity, shows more than 20-fold higher efficiency towards ChoKα1 than ChoKβ. Conclusion/Significance This study represents the first evidence of the distinct metabolic role of ChoKα and ChoKβ isoforms, suggesting different physiological roles and implications in human carcinogenesis. These findings constitute a step forward in the design of an antitumoral strategy based on ChoK inhibition.This work has been supported by grants to JCL from Comunidad de Madrid (GR-SAL-0821-2004), Ministerio de Ciencia e Innovación (SAF2008-03750, RD06/0020/0016), Fundación Mutua Madrileña, and by a grant to ARM from Fundación Mutua Madrileña.Peer reviewe

Serum and tissue profiling in bladder cancer combining protein and tissue arrays

Aiming at identifying biomarkers for bladder cancer, the serum proteome was explored in a pilot study through a profiling approach using protein arrays. Supervised analyses identified a panel 171 immunogenic proteins differentially expressed between patients with bladder cancer (n = 12) and controls without the disease (n = 10). The microanatomical expression patterns of novel immunogenic proteins, especially dynamin and clusterin, were found significantly associated with histopathologic variables and overall survival, as confirmed by immunohistochemistry using an independent series of bladder tumors contained in tissue microarrays (n = 289). Thus, the protein arrays approach has identified a panel of immunogenic candidates that may potentially play a role as diagnostic biomarkers, especially for muscle invasive disease. Moreover, the protein expression patterns of dynamin and clusterin in bladder tumors were shown to adjunct for histopathologic staging and clinical outcome prognosisThis work was supported by a grant to Dr. Sánchez-Carbayo from the Spanish Ministry of Education and Science (SAF2006-08519). Esteban Orenes and Rodrigo Barderas are recipient of Postdoctoral Contracts of the FIS supported by the Spanish Ministry of HealthPeer reviewe

Comparative expression of ChoKα1 and ChoKβ1 mRNA in a panel of cancer cell lines.

<p>Q-PCR was performed to determine the level of expression of mRNA in non-tumorogenic mammary cell lines (HMEC, MCF10A), breast cancer cell lines (MDA-MB435, MDA-MB468, T47D, MCF7), non-tumorogenic lung cells (BEC) and lung cancer cell lines (H510, H82). The data were normalized with the endogenous 18S ribosomal RNA. For the comparison between tumoral and non-tumoral cell lines, the 2^−ΔΔCt method was applied and log₁₀ RQ is represented. Note that the data are referred to the Human Mammary Epithelial Cells (HMEC) mRNA levels in breast cell lines and no significant difference in the level of both ChoK isoforms mRNA was found with the normal MCF10A cell line. The reference for lung cancer cells was the primary Bronchial Epithelial Cells (BEC).</p

Overexpression of ChoKβ1 is not sufficient to induce tumor growth in athymic nude mice.

<p>Xenografts were established by s.c. injection of transfected HEK293T or MDCK cells in athymic nu/nu nude mice. <b>A)</b> and <b>B)</b> Western Blot analysis of ectopic expression of choline kinase isoforms in transfected HEK293T or MDCK cells, respectively, before mice inoculation. <b>C)</b> and <b>D)</b> Analysis of choline kinase activity in ChoKα1 or β1 transfected HEK293T or MDCK cells-free extracts before mice inoculation. <b>E)</b> and <b>F)</b> Volume of tumors generated by subcutaneous injection of 10⁶ transfected cells. Tumoral volume was calculated according to the formula: <i>Vol = [D * d²]/2</i>, where D and d are major and minor tumor diameters respectively. The data from HEK293T represents mean values±SEM from two independent experiments (n₁ = 12; n₂ = 16), the MDCK experiment correspond to an equivalent experiment with n = 12.</p

Anchorage independent cell growth of ChoKα1- and β1-overexpressing cells.

<p><b>A)</b> and <b>B)</b><i>In vitro</i> ChoK activity of cell-free extracts from transfected cells at the moment of plating, determined as conversion of ¹⁴C-labeled choline to PCho. <b>C)</b> Photographs of a representative experiment of the soft agar assay. A total of 10⁵ cells were plated per 60-mm dish, and the number of colonies quantified after 5-8 weeks of incubation. <b>D)</b> and <b>E)</b> Computer based automatic quantification of the number of colonies, mean values±SEM is represented. The assay was performed 3 independent times with triplicate samples obtaining similar results. Statistical significance (p≤0.05) is marked by an asterisk.</p

Differential activation of choline kinase α1 and β1 isoforms by Ras and Rho GTPases.

<p>Choline kinase isoforms were expressed alone or in combination with the indicated Ras and Rho GTPases and the <i>in vitro</i> ChoK activity determined. <b>A)</b> Analysis of ectopic expression by Western Blot in HEK293T transfected cell extracts of ChoKα1 (52 KDa), ChoKβ1(45 KDa), RhoA-QL(22 KDa), Cdc42-QL(25 KDa) and H-rasV12 (23 KDa). Empty vectors were used as controls for the endogenous levels, and GAPDH as loading control. <b>B)</b> and <b>C)</b><i>In vitro</i> choline kinase activity of ChoKα1 or ChoKβ1 in the presence of enhanced expression of constitutive active forms of RhoA, Cdc42 or H-Ras. <b>D)</b> and <b>E)</b><i>In vitro</i> ethanolamine kinase activity of ChoKα or ChoKβ in the presence of each indicated constitutive active form of GTPase. The results are represented as fold induction of conversion to the corresponding phosphorylated metabolite determined as total cpm/µg of whole cellular extract, and normalized to the empty vector transfected cells as control. Data shown represent the mean values±SEM of 3 independent experiments, each one performed with duplicate samples. Statistical significance (p≤0.05) is marked by an asterisk comparing to the activity achieved when ChoKα1 or ChoKβ1, where appropriate, are transfected alone.</p

Michaelis constant (Km) of ChoKα and β isoforms for choline and ethanolamine.

1<p>Data are represented in milliMolar.</p>2<p>Referenced to the lowest Km.</p><p>Km of the different isoforms of ChoK for each substrate is indicated in each case. The results were obtained from four independent experiments using the logarithmic Michaelis-Menten formula as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007819#s4" target="_blank">Material and Methods</a>.</p

Characterization of choline and ethanolamine kinase activity of ChoKα1 and Chokβ1 in HEK293T cells.

<p>HEK293T cells were transfected with eukaryotic expression vectors of human ChoKα1 and ChoKβ1 gene. pCDNA3b empty vector was used as control. <b>A)</b> Overexpression of ChoKα1 and ChoKβ1 in HEK293T cells detected by Western Blot. GAPDH detection was used as control of expression level. <b>B, C)</b> In vitro ChoK (B) and EtnK (C) activity of choline kinase α1 and β1 isoforms in cell-free extracts of HEK293T transfected cells. Percentage of conversion of ¹⁴C-choline or ¹⁴C-ethanolamine to the phosphorylated product is represented. The experiment was performed in duplicate samples, repeated 4 times, and mean±SEM values from all experiments estimated.</p