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CALCIUM RELEASE-ACTIVATED CALCIUM INFLUX IN CULTURED HUMAN MESANGIAL CELLS
No full textCa2+ influx is a major component of the response of cultured human mesangial cells (HMC) to vasoconstrictors. Activators of phospholipase C such as angiotensin II (Ang II) release Ca2+ from intracellular stores and enhance Ca2+ influx, which in turn is modulated by Na+/Ca2+ exchange. By microfluorometry we studied the mechanisms of Ca2+ entry in resting and stimulated fura-2-loaded monolayers or single HMC. Addition of 1 to 10 mM extracellular Ca2+ to cells equilibrated in Ca2+-free media resulted in a rapid, persistent elevation of free cytosolic Ca2+ ([Ca2+](i)), from 52 +/- 5 to 113 +/- 18 and 226 +/- 37 nM, respectively. Ca2+ influx was blocked by lanthanum or chelation with EGTA, while it was only partially inhibited by voltage-operated Ca2+ channel(VOC) blockers, such as nifedipine or verapamil. The rise of [Ca2+](i) at high external [Ca2+] was not due to a Ca2+-sensing mechanism with release of intracellularly stored Ca2+, since it was prolonged, and it was not seen in cells maintained in normal 1.25 mM [Ca2+] media. Moreover, it was not abolished by prior depletion of Ca2+ stores with 0.5 mu M thapsigargin or 5 mu M ionomycin in Ca2+-free media, which transiently increased [Ca2+](i) (to 281 +/- 39 and 380 +/- 51 nM, respectively). On the contrary, both agents markedly potentiated Ca2+ influx upon addition of 1 to 10 mM [Ca2+](c), (to a maximum of 686 +/- 111 and 633 +/- 150 nM, P < 0.05 vs. control). Prior stimulation of [Ca2+](i) with 1 mu M Ang II had similar effects, enhancing the subsequent Ca2+ influx to 241 +/- 42 (1 mM Ca2+) and 512 +/- 106 nM (10 mM Ca2+; P < 0.05). Enhancement of Ca2+ influx by thapsigargin, ionomycin and Ang II was confirmed by increased Mn2+ quenching of fura-2 fluorescence following addition of the agents in the absence of extracellular Ca2+. VOC activation by membrane depolarization was not responsible for such potentiation, since 50 mM KCl failed to modify Ca2+ influx. Na+/Ca2+ exchange was ruled out by persistence of influx after intracellular Na+ depletion. Thus, an initial elevation of [Ca2+](i) by vasoconstrictors, blockers of Ca2+ ATPase or Ca2+ ionophores enhances Ca2+ entry in HMC via a Ca2+ release-activated Ca2+ conductance, mostly independent of plasma membrane depolarization
Effects of advanced glycation end products on cytosolic Ca2+ signaling of cultured human mesangial cells
No full textAdvanced glycation end product (AGE) accumulation in a high glucose (HG) environment is thought to mediate some of the vascular complications of diabetes. Transmembrane signaling of contractile cells is generally inhibited by HG, with implications for systemic and target organ hemodynamics. In the kidney, glomerular mesangial cells grown in HG media are hyporesponsive to the effects of vasoconstrictor agents, possibly explaining the hyperfiltration and increased capillary pressure that eventually lead to diabetic glomerulopathy. To verify whether AGE binding to specific mesangial receptors could mediate these effects of HG, cultured human mesangial cells (HMC) were exposed to in vitro glycated bovine serum albumin (BSA) for 60 min at 37 degrees C before measurement of cytosolic Ca2+ ([Ca2+](i)) by microfluorometric techniques in monolayers or single cells. AGE-BSA (2 mg/ml) reduced Ca2+ release from intracellular stores by 1 mu M angiotensin II from peak [Ca2+](i) levels of 843 +/- 117 to 390 +/- 50 nM in monolayers and from 689 +/- 68 to 291 +/- 36 nM in individual cells (P < 0.05). Nonglycated BSA and BSA exposed to 250 mM glucose-6-phosphate for 30 d in the presence of 250 mM aminoguanidine (AMGD), an inhibitor of nonenzymatic glycation, had no effect on the angiotensin II-induced [Ca2+](i) spike (peak 766 +/- 104 and 647 +/- 87 nM, monolayers/ single cells, respectively, P = NS). AGE also inhibited store-operated Ca2+ influx through plasma membrane channels, assessed by addition of 1 to 10 mM extracellular Ca2+ to cells previously held in Ca2+-free media (control 339 +/- 46/593 +/- 51, +AGE-BSA 236 +/- 25/390 +/- 56, +AMGD 483 +/- 55/ 374 +/- 64 nM [Ca2+](i) monolayers/single cells at 10 mM Ca2+, respectively; +AGE-BSA, P < 9.05 versus control). Contrary to PIG, AGE-BSA did not translocate protein kinase C isoforms alpha, zeta, and delta to the plasma membrane. Culture of HMC in HG supplemented with 1 mM AMGD prevented downregulation of [Ca2+](i) signaling. These data suggest that glycated macromolecules or matrix components may inhibit transmembrane Ca2+ signaling of glomerular cells through binding to a specific AGE receptor, thus mediating some of the known functional effects of HG on the kidney
Monocyte/mesangial cell interactions in high-glucose co-coltures
No full textBACKGROUND:
Monocytes bind to human mesangial cells (HMC) in a co-culture model of leukocyte/ glomerular cell interactions. Since monocytic infiltration has been demonstrated in the early stages of diabetic glomerulopathy, we examined whether co-culture with myelomonocytes of the U937 cell line in media mimicking the diabetic microenvironment modulated phenotype, growth, and extracellular matrix production patterns of HMC.
METHODS:
HMC monolayers grown for 5 days in 5.5 mmol/l (NG) or 30 mmol/l (HG) glucose media were examined 3, 24 and 48 h after addition of U937 cells by computer-assisted image analysis/fluorescence microscopy following fixation, staining for cell adhesion, and TUNEL/propidium iodide labelling for apoptosis. As matrix components may be relevant to both phenotype of cultured HMC and monocyte adhesion, reverse transcription-polymerase chain reaction, zymography, and ELISA were used to detect urokinase-plasminogen activator (uPa), collagen type IV (COL IV), transforming growth factor beta1 (TGF-beta1), matrix metalloproteinases (MMP), and relative inhibitors (tissue inhibitor of MMP (TIMP)) expression in co-cultures in NG/HG.
RESULTS:
U937 adhesion at 1-3 h was increased in HG (from 54.9+/-6.6 to 87.1+/-5.8% U937/HMC). Control HMC proliferating in NG supplemented with 10% fetal bovine serum had an average cross-sectional area of 9993+/-505 micro(2) with 1.2+/-0.1 hillocks/high-power field, which increased to 13 651+/- 1114 micro(2) with 0.5+/-0.2 hillocks/high-power field in HG (P<0.05). TUNEL+HMC were nearly identical (4.9+/-1.7 vs 4.2+/-0.4% in HG, P=NS). Enhanced transcription and secretion of urokinase (uPA, +656%), COL IV (+137%), TGF-beta1 (+590%) were observed in co-cultures in HG. COL IV and TGF-beta1, but not uPA, were also increased in HMC alone, exposed to HG for 5 days. MMP-2/TIMP-2 ratio was decreased while MMP-1/TIMP-1 was increased in HG co-cultures. In both NG and HG, U937 adhesion reduced HMC number and hillocks at 24 h, with constant apoptosis. The effects of U937 were no longer detectable at 48 h, when apoptosis was 2.1+/-0.6 vs 4.0+/-0.4% in HG, and cell counts returned above basal, possibly due to a delayed proliferative response.
CONCLUSIONS:
High glucose medium increases U937 cell adhesion to HMC. In turn, monocytes modulate number and spatial distribution of HMC, which are also markedly affected by ambient glucose levels. These interactions may be relevant to leukocyte infiltration, mesangial expansion, and glomerulosclerosis in diabetes
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Changes in bone turnover after parathyroidectomy in dialysis patients: role of calcitriol administration.
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Two-site immunoradiometric intact parathyroid hormone assay versus C-terminal parathyroid hormone in predicting osteodystrophic bone lesions in predialysis chronic renal failure.
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