Thin-basement-membrane nephropathy (TBMN) and Alport syndrome (AS) are progressive collagen IV nephropathies caused by mutations in COL4A3/A4/A5 genes. These nephropathies invariably present with microscopic hematuria and frequently progress to proteinuria and CKD or ESRD during long-term follow-up. Nonetheless, the exact molecular mechanisms by which these mutations exert their deleterious effects on the glomerulus remain elusive. We hypothesized that defective trafficking of the COL4A3 chain causes a strong intracellular effect on the cell responsible for COL4A3 expression, the podocyte. To this end, we overexpressed normal and mutant COL4A3 chains (G1334E mutation) in human undifferentiated podocytes and tested their effects in various intracellular pathways using a microarray approach. COL4A3 overexpression in the podocyte caused chain retention in the endoplasmic reticulum (ER) that was associated with activation of unfolded protein response (UPR)-related markers of ER stress. Notably, the overexpression of normal or mutant COL4A3 chains differentially activated the UPR pathway. Similar results were observed in a novel knockin mouse carrying the Col4a3-G1332E mutation, which produced a phenotype consistent with AS, and in biopsy specimens from patients with TBMN carrying a heterozygous COL4A3-G1334E mutation. These results suggest that ER stress arising from defective localization of collagen IV chains in human podocytes contributes to the pathogenesis of TBMN and AS through activation of the UPR, a finding that may pave the way for novel therapeutic interventions for a variety of collagenopathies.

Micro-RNAs (miRNAs) play a pivotal role in the development and physiology of the cardiovascular system while they have been associated with multiple cardiovascular diseases (CVDs). Several cardiac miRNAs are detectable in circulation (circulating miRNAs; c-miRNAs) and are emerging as diagnostic and therapeutic biomarkers for CVDs. c-miRNAs exhibit numerous essential characteristics of biomarkers while they are extremely stable in circulation, their expression is tissue-/disease-specific, and they can be easily detected using sequence-specific amplification methods. These features of c-miRNAs are helpful in the development of non-invasive assays to monitor the progress of CVDs. Despite significant progress in the detection of c-miRNAs in serum and plasma, there are many contradictory publications on the alterations of cardiac c-miRNAs concentration in circulation. The aim of this review is to examine the pre-analytical and analytical factors affecting the quantification of c-miRNAs and provide general guidelines to increase the accuracy of the diagnostic tests in order to improve future research on cardiac c-miRNAs.

AND-34, a novel GDP exchange factor, is expressed constitutively at significant levels in murine splenic B cells, but not in murine splenic T cells or thymocytes. In B cell lines, anti-IgM treatment up-regulates AND-34 transcript levels. B cell AND-34 associates with both the docking molecules p130Cas and HEF1. AND-34 binds by its GDP exchange factor domain to the C terminus of HEF1, a region of HEF1 previously implicated in apoptotic, adhesion, and cell cycle-regulated signaling. Overexpression of AND-34 in murine B cell lines activates the Rho family GTPase Cdc42, but not Rac, Rho, RalA, or Rap1. Consistent with this, a subpopulation of AND-34 overexpressing B cells have long filamentous actin-containing cellular extensions. AND-34 overexpression augments both autophosphorylation and kinase activity of the Cdc42/Rac-responsive serine/threonine kinase PAK1. As previously reported for lymphoid cells transfected with constitutively active Cdc42, AND-34 overexpression inhibits SDF-1α-induced B cell polarization. These studies suggest that p130Cas and HEF1-associated AND-34 may regulate B cell adhesion and motility through a Cdc42-mediated signaling pathway.

IntroductionCyclic nucleotide signaling in lymphoid cells is regulated by a diverse set of phosphodiesterase (PDE) families, and selective inhibitors of these PDEs have proven to be both of therapeutic interest and of use in the analysis of lymphoid signal transduction pathways. At a minimum, lymphoid cells express PDE1B, PDE3B, PDE4A, B, and D, and PDE7A; all enzymes that can catabolize 3Ј:5Ј cyclic adenosine monophosphate (cAMP). [1][2][3][4][5] In studies of the mature B-cell malignancy B-cell chronic lymphocytic leukemia (B-CLL), we and others have found that both nonspecific PDE inhibitors such as theophylline and the PDE4-specific inhibitor rolipram induce apoptosis in leukemic cells over a 48-to 72-hour period. [6][7][8] While rolipram also induces apoptosis in peripheral B cells, albeit superimposed on a high normal basal apoptotic rate, the same agent has little or no apoptotic effect on peripheral T cells. 8 PDE4 inhibitors induce a mitochondrial apoptotic pathway in B-CLL cells with release of cytochrome c, caspase 9 and 3 activation, and PARP (poly-adenosine diphosphate [ADP] ribose polymerase) cleavage. This pathway may be triggered by PDE4 inhibitor-induced up-regulation of serine/threonine phosphatase protein phosphatase 2A (PP2A) activity with resultant dephosphorylation of the proapoptotic BH3-only Bcl2 family member BAD, release of BAD from the cytosolic adapter protein 14-3-3, and translocation of BAD to mitochondria. 9 In contrast, although PDE3B is expressed in B-CLL, when used alone PDE3 inhibitors do not induce apoptosis in B-CLL. 10 PDE7A is expressed and regulated in B-CLL cells, but reagents that allow selective inhibition of this PDE over the 48 to 72 hours required for apoptosis studies have not yet become available. 11 While most studies of cAMP signaling in lymphoid cells have focused on protein kinase A (PKA), recently a novel family of cAMP effector proteins, exchange protein directly activated by cAMP 1 (EPAC1) and EPAC2 (also known as cAMP-guanine nucleotide exchange factor I/II [GEFI/II]) has been identified in other cell lineages. 12-14 Upon binding cAMP, EPAC catalyzes release of guanosine 5Ј-diphosphate (GDP) from the Ras family guanosine 5Ј-triphosphatases (GTPases) Rap1 and Rap2, allowing the more abundant intracellular GTP to bind and convert the GTPase to an active, signaling conformation. 12 In an effort to develop a reagent that would allow selective analysis of EPACinduced signaling, Enserink et al 15 subsequently identified a cAMP analog, 8CPT-2Me-cAMP (8-(4-chloro-phenylthio)-2Ј-O-methyladenosine-3Ј,5Ј-cAMP), that activates EPAC while having little or no activity on PKA. While prior studies implicated Rap1 in a lineage-dependent manner in either cAMP-induced activation or inhibition of extracellular signal-regulated kinase (ERK), experiments using 8CPT-2Me-cAMP failed to support a role for EPAC or The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisem...