Autosomal-dominant polycystic kidney disease is a multiorgan disease and its vascular manifestations are common and life-threatening. Despite this, little is known about their pathogenesis. Somatic mutations to the normal PKD allele in cystic epithelia and cyst development associated with the unstable Pkd2(WS25) allele suggest a two-hit model of cystogenesis. However, it is unclear if this model can account for the cardiovascular pathology or if haploinsufficiency alone is disease-associated. In the present study, we found a decreased polycystin-2 (PC2, protein encoded by Pkd2 gene) expression in Pkd2( +/-) vessels, roughly half the wild-type level, and an enhanced level of intracranial vascular abnormalities in Pkd2 (+/-) mice when induced to develop hypertension. Consistent with these observations, freshly dissociated Pkd2 (+/-) vascular smooth muscle cells have significantly altered intracellular Ca(2+) homeostasis. The resting [Ca(2+)](i) is 17.1% lower in Pkd2 (+/-) compared with wild-type cells (P=0.0003) and the total sarcoplasmic reticulum Ca(2+) store (emptied by caffeine plus thapsigargin) is decreased (P<0.0001). The store operated Ca(2+) (SOC) channel activity is also decreased in Pkd2 (+/-) cells (P=0.008). These results indicate that inactivation of just one Pkd2 allele is sufficient to significantly alter intracellular Ca(2+) homeostasis, and that PC2 is necessary to maintain normal SOC activity and the SR Ca(2+) store in VSMCs. Based on these findings, and the fact that [Ca(2+)](i) signaling is essential to the regulation of contraction, production and secretion of extracellular matrix, cellular proliferation and apoptosis, we propose that the abnormal intracellular Ca(2+) regulation associated with Pkd2 haploinsufficiency is directly related to the vascular phenotype.

Research has found that downsizing is not generally followed by improved organizational performance. Using a sample of hospitals that recently downsized, we evaluate the effects on performance of the human resource management (HRM) practices used in layoffs. Strategic HRM theory suggests that practices can have an impact on performance outcomes. We find that showing consideration for employees' morale and welfare during downsizing is positively related both to perceived success of downsizing and to financial performance following layoffs. Advance notice of layoffs is positively related to subsequent financial performance, but the provision of extended insurance to laid‐off employees is negatively related to financial performance. Planned redesign of work structures is positively related to perceived success, but has neutral to negative effects on financial performance. Copyright © 2004 John Wiley & Sons, Ltd.

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Effects of hypothyroidism on maximum specific force in rat diaphragm muscle fibers. J Appl Physiol 92: 1506-1514, 2002.. First published January 7, 2002 10.1152/japplphysiol.00095.2001.-We hypothesized that 1) hypothyroidism (Hyp) decreases myosin heavy chain (MHC) content per half-sarcomere in diaphragm muscle (Diam) fibers, 2) Hyp decreases the maximum specific force (Fmax) of Diam fibers because of the reduction in MHC content per half-sarcomere, and 3) Hyp affects MHC content per half-sarcomere and Fmax to a greater extent in fibers expressing MHC type 2X (MHC2X) and/or MHC type 2B (MHC2B). Studies were performed on single Triton X-permeabilized fibers activated at pCa 4.0. MHC content per half-sarcomere was determined by densitometric analysis of SDS-polyacrylamide gels and comparison with a standard curve of known MHC concentrations. After 3 wk of Hyp, MHC content per half-sarcomere was reduced in fibers expressing MHC2X and/or MHC2B. On the basis of electron-microscopic analysis, this reduction in MHC content was also reflected by a decrease in myofibrillar volume density and thick filament density. Hyp decreased Fmax across all MHC isoforms; however, the greatest decrease occurred in fibers expressing fast MHC isoforms (ϳ40 vs. ϳ20% for fibers expressing slow MHC isoforms). When normalized for MHC content per half-sarcomere, force generated by Hyp fibers expressing MHC2A was reduced compared with control fibers, whereas force per half-sarcomere MHC content was higher for fibers expressing MHC2X and/or MHC2B in the Hyp Diam than for controls. These results indicate that the effect of Hyp is more pronounced on fibers expressing MHC 2X and/or MHC2B and that the reduction of Fmax with Hyp may be at least partially attributed to a decrease in MHC content per half-sarcomere but not to changes in force per cross bridge. thyroid hormone levels; skinned fibers; myosin heavy chain content; force per cross bridge MYOSIN HEAVY CHAIN (MHC) isoform expression forms the basis of fiber type classification in skeletal muscle, and contractile characteristics of skeletal muscle are correlated with MHC isoform expression (3,37,40,43,44). Thyroid hormone has been shown to modulate MHC gene expression and isoform composition in adult skeletal muscle fibers (10,13,22,29,30,32,34,51). However, the effects of thyroid hormone in the rat diaphragm muscle (Dia m ) are controversial. For example, previous studies have shown that hypothyroidism (Hyp) has no effect on the rat Dia m (29), whereas others have shown an increase in the relative number of type I Dia m fibers and a decrease in Dia m oxidative enzyme capacity (28). Previous results from our laboratory demonstrated changes in relative MHC isoform composition, a decrease in maximum specific force (F max ), and a slowing of maximum shortening velocity in adult Dia m bundles after 3 wk of Hyp (22).Studies from our laboratory and others indicate that the F max of single fibers differs with MHC isoform expression (3,18,19,43,44). Dia m fibers expressing MHC type 2X (MHC 2X ) alone...

Vascular complications are the leading cause of morbidity and mortality in autosomal dominant polycystic kidney disease. Although evidence suggests an abnormal vascular reactivity, contractile function in Pkd mutant vessels has not been studied previously. Contractile response to phenylephrine (PE; 10 ؊10 to 10 ؊4 M), an ␣1-adrenergic receptor agonist, was examined.De-endothelialized Pkd2 ϩ/Ϫ aortic rings generated a higher maximum force (

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Abstract-CardiovascularKey Words: cAMP Ⅲ phosphodiesterase Ⅲ proliferation Ⅲ ERK Ⅲ apoptosis A utosomal-dominant polycystic kidney disease (ADPKD) is caused by mutations to either PKD1 or PKD2 genes encoding polycystin-1 (PC1) or polycystin-2 (PC2), respectively. PC1 is a plasma membrane receptor-like protein that has functions in cell-cell or cell-extracellular matrix interactions. PC2 is a nonselective cation channel protein with a large single-channel conductance and high permeability to calcium (Ca 2ϩ ). PC1 interacts with PC2. Their interaction can form a functional receptor-ion-channel-complex and regulate heterotrimeric G-protein-mediated signaling cascades. The mutations of either gene cause a nearly identical clinical phenotype (see review 1 ).Cardiovascular complications are the leading cause of mortality and morbidity in ADPKD. The incidence of intracranial aneurysms/aneurysmal ruptures and thoracic aortic dissections in ADPKD is approximately 10-fold higher than in the general population. 2 Even during the early stages of ADPKD, before the onset of hypertension or renal dysfunction, abnormal thickening of the intrarenal arteries and reduction in renal blood flows are evident. 3,4 Although clinical and experimental evidence indicate a close relation between PKD/Pkd mutations and vascular complications, their pathogenesis is not understood. We have shown that when induced to develop hypertension, Pkd2 ϩ/Ϫ mice have an increased susceptibility to vascular injury, manifested as premature death or developing prominent irregular thickening in the tunica media layer of intracranial vessels. 5 The areas of irregular vessel wall thickening are correlated with abnormally increased or decreased number of VSMCs, indicating an imbalance between smooth muscle cellular proliferation and apoptosis.Elevated rates of proliferation and apoptosis are the major phenotypic features of ADPKD cells; these abnormalities have been detected in multiple organ systems including kidneys, lungs, liver, heart, brain, spleen, thymus, and testis. 6,7 Recent studies suggest that reduced basal intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) and elevated intracellular cyclic 3Ј:5Ј-adenosine monophosphate (cAMP) play a role in this phenotype. Cystic renal tissues have elevated intracellular cAMP. 8,9 In contrast to its growth inhibitory effect on wild-type renal epithelial cells, cAMP promotes proliferation in PKD/Pkd mutant renal epithelial cells. 10 -12 This proliferative response to cAMP can also be induced in wild-type renal epithelial cells by reducing their basal [Ca 2ϩ ] i , 13 indi-

Calcifying biologic nanoparticles (NPs) develop under cell culture conditions from homogenates of diverse tissue samples displaying extraosseous mineralization, including kidney stones and calcified aneurysms. Probes to definitively identify NPs in biologic systems are lacking. Therefore, the aim of this study was to begin to establish a proteomic biosignature of NPs in order to facilitate more definitive investigation of their contribution to disease. Biologic NPs derived from human kidney stones and calcified aneurysms were completely decalcified by overnight treatment with EDTA or brief incubation in HCl, as evidenced by lack of a calcium shell and of Alizarin Red S staining, by transmission electron microscopy and confocal microscopy, respectively. Decalcified NPs contained numerous proteins including some from bovine serum and others of prokaryotic origin. Most prominent of the latter group was EF-Tu, which appeared identical to EF-Tu from S. epidermidis. A monoclonal antibody against human EF-Tu recognized a protein in Western blots of total NP lysate, as well as in intact NPs by immunofluorescence and immunogold EM. Approximately 8% of NPs were quantitatively recognized by the antibody by flow cytometry. Therefore, we have defined methods to reproducibly decalcify biologic NPs, and identified key components of their proteome. These elements, including EF-Tu, can be used as biomarkers to further define processes which mediate propagation of biologic NPs and their contribution to disease.