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D S Emmanouel

Publications and source records attributed to D S Emmanouel.

At least 19 recordsLinked to original sources
PubMed2000

PLC-gamma1 signaling pathway and villin activation are involved in actin cytoskeleton reorganization induced by Na+/Pi cotransport up-regulation.

BACKGROUND: The brief incubation of opossum kidney (OK) cells with low P(i) results in Na+/P(i) cotransport up-regulation and in substantial, but transient, cytoskeletal reorganization. In this study, we examined signaling events involved in the depolymerization of microfilaments. RESULTS: Confocal laser scanning microscopy, immunoblot and immunoprecipitation experiments revealed villin co-localization with mainly actin short filaments and monomers, indicating that under the conditions used, villin acted as an actin-severing protein. Further analysis revealed that low concentrations of extracellular phosphate resulted in phospholipase Cgammal (PLC-gammal) translocation to the actin cytoskeleton, without increases in its tyrosine phosphorylation. Additionally, tyrosine phosphorylation of a portion of insoluble villin was increased; whereas, only tyrosine phosphorylated villin associated with PLC-gammal. Although, tyrosine phosphorylation of PLC-gammal was not observed during Na+/P(i) cotransport up-regulation, genistein treatment abolished the enzyme's translocation to the actin cytoskeleton, as well as its association with villin. In addition, villin was found to associate with the 85-KDa subunit (p85) of phosphatidylinositol (PI)-3 kinase, concomitant with PLC-gammal, in the cytoskeletal fraction of Na+/P(i) cotransport up-regulated cells. CONCLUSIONS: Our observations suggest a signaling mechanism linking low ambient P(i) levels to the acute up-regulation of its cotransport with sodium and the depolymerization of the subcortical actin cytoskeleton.

Actin Cytoskeleton↗
PubMed1999

Acute onset of dermatomyositis presenting in pregnancy with rhabdomyolysis and fetal loss.

We report a case of acute onset of dermatomyositis with rhabdomyolysis and myoglobinuria, which presented in the 14th week of pregnancy and resulted in spontaneous abortion of the fetus. The diagnostic work up for an underlying disease was negative and the histologic examination confirmed the diagnosis of dermatomyositis, which subsequently improved with corticosteroids.

Abortion, Spontaneous↗
PubMed1999

Pneumothorax complicating fatal bronchiolitis obliterans organizing pneumonia.

Bronchiolitis obliterans organizing pneumonia (BOOP) is an uncommon pulmonary disorder, the clinical spectrum of which is variable. We present a fatal case of BOOP, which developed spontaneous pneumothorax, a complication considered rare. Unusual was also the upper lobe distribution of the infiltrates. The histologically diagnosed disease failed to respond to antibiotics and corticosteroids and the 74-year-old patient eventually succumbed with acute respiratory distress syndrome, 50 days after disease onset. Spontaneous pneumothorax should be added to the complications of BOOP, which may adversely affect prognosis.

Aged↗
PubMed1999

TNF-alpha induces actin cytoskeleton reorganization in glomerular epithelial cells involving tyrosine phosphorylation of paxillin and focal adhesion kinase.

Glomerular permeability for macromolecules depends partially on proper attachment of the glomerular epithelial cells (GEC) to the glomerular basement membrane (GBM). The latter requires integrity of the actin cytoskeleton, which in turn is regulated by specific actin-associated proteins. Since several glomerulopathies characterized by heavy proteinuria are associated with increased glomerular tumor necrosis factor alpha (TNF-alpha) expression, we studied the interaction of TNF-alpha with the actin cytoskeleton of cultured rat GEC. Incubation of GEC with 10 ng/ml TNF-alpha for variable time periods ranging from 15 min to 24 hr demonstrated a marked accentuation and redistribution of actin microfilaments, as shown by direct fluorescence analysis and confocal laser scanning microscopy. Quantitative biochemical determination of the G/total-actin ratio confirmed the above observations. Indeed, this ratio was significantly reduced, indicating substantial polymerization of G-actin and formation of F-actin. Concurrently, TNF-alpha rapidly induced tyrosine phosphorylation of both paxillin and focal adhesion kinase, without affecting the expression levels of these two proteins. In addition, tyrosine phosphorylation of vinculin became evident, indicating involvement of this focal adhesion marker in the observed actin reorganization. Inhibition of tyrosine phosphorylation by genistein prevented the reorganization of the actin cytoskeleton by TNF-alpha. We conclude that TNF-alpha induces substantial reorganization of actin cytoskeleton and focal adhesions. These effects occur simultaneously, with a prompt TNF-alpha-induced tyrosine phosphorylation of paxillin and focal adhesion kinase, indicating that these proteins, known to regulate actin polymerization and formation of focal adhesions, may be directly involved in the mechanism controlling the observed actin redistribution. These findings suggest that the observed TNF-alpha-actin cytoskeleton interactions may relate to the pathogenesis of glomerulopathies with heavy proteinuria, in which increased glomerular expression of TNF-alpha is associated with disturbances in the attachment of podocytes to the GBM.

Actins↗
PubMed1998

Early alterations of actin cytoskeleton in OK cells by opioids.

Recently we identified and characterized opioid binding sites in OK (opossum kidney) cells and observed decreased proliferation of these cells in response to opioids. In the present study we investigated the effects of opioids on the actin cytoskeleton and explored whether their antiproliferative action may relate to alterations in the distribution or the dynamics of actin microfilaments. Exposure of OK cells to the opioids alphaS1 casomorphin and ethylketocyclazocine resulted in a rapid and substantial actin microfilament reorganization. This was documented by a significant dose-dependent decrease in the amounts of F-actin, determined by measurements of quantitative fluorescence, by immunoblot analysis and by a concomitant increase of the G/total-actin ratio measured by the DNase I inhibition assay. These changes were verified by confocal laser scanning microscopy, which showed marked redistribution of the microfilamentous structures in the presence of the opioids without affecting the organization of microtubules or vimentin intermediate filaments. The effect of opioids on actin polymerization dynamics occurred within 15 min and persisted for at least 2 h, while their restoration to control levels was accomplished 6 h later, indicating a reversible phenomenon. Northern blot analysis showed that the concentration of the actin transcript was unaffected. The addition of diprenorphine, a general opioid antagonist, prevented the effects of opioids on the actin cytoskeleton. The inhibition of OK cell proliferation, induced by ethylketocyclazocine and alphaS1 casomorphin was partially prevented in the presence of phallacidin, which stabilizes microfilaments. Our findings demonstrate that opioids, acting via kappa 1 binding sites, induce rapidly modifications in the dynamics of actin polymerization, and in the organization of microfilaments in OK cells, which may relate to their antiproliferative effect on these cells.

Actins↗
PubMed1996

Identification and characterization of opioid and somatostatin binding sites in the opossum kidney (OK) cell line and their effect on growth.

Opioids and somatostatin analogs have been implicated in the modulation of renal water handling, but whether their action is accomplished through central and/or peripheral mechanisms remains controversial. In different cell systems, on the other hand, opioids and somatostatin inhibit cell proliferation. In the present study, we have used an established cell line, derived from opossum kidney (OK) proximal tubules, in order to characterize opioid and somatostatin receptors and to investigate the action of opioids and somatostatin on tubular epithelial tissue. Our results show the presence of one class of opioid binding sites with kappa, selectivity (KD 4.6 +/- 0.9 nM, 57,250 sites/cell), whereas delta, mu, or other subtypes of the kappa site were absent. Somatostatin presents also a high affinity site on these cells (KD 24.5 nM, 330,000 sites/cell). No effect of either opioids or somatostatin on the activity of the NA+/Pi cotransporter was observed, indicating that these agents do not affect ion transport mechanisms. However, opioid agonists and somatostatin analogs decrease OK cell proliferation in a dose-dependent manner; in the same nanomolar concentration range, they displayed reversible specific binding for these agents. The addition of diprenorphine, a general opioid antagonist, reversed the effects of opioids, with the exception of morphine. Furthermore, morphine interacts with the somatostatin receptor in this cell line too, as was the case in the breast cancer T47D cell line. Our results indicate that in the proximal tubule opioids and somatostatin do not affect transport, but they might have a role in the modulation of renal cell proliferation either during ontogenesis or in kidney repair.

Animals↗
PubMed1996

Na+/Pi co-transport alters rapidly cytoskeletal protein polymerization dynamics in opossum kidney cells.

We studied with biochemical and immunofluorescent techniques the interactions between the actin microfilament and tubulin microtubule cytoskeleton and Na+/P1 co-transport in opossum kidney cells, a line with proximal tubular characteristics. On brief (5 min) incubation of the cells with a low (0.1 mM) concentration of Pi, a rapid F-actin depolymerization takes place, which fails to occur in cells incubated under similar conditions with 1 mM Pi. The disassembly of actin microfilaments could be quantitatively expressed as a 33% increase in the ration of monomeric G-actin to polymerized F-actin (G/F-actin ration from 0.80 +/- 0.03 to 1.06 +/- 0.06, n = 28, P<0.01), owing to a significant decrease in the latter. Under these conditions microfilaments were also markedly destabilized, as shown by their diminished resistance to graded cytochalasin B concentrations. In addition, incubation of opossum kidney cells with low Pi concentrations (0.1 mM) resulted within 5 min in a substantial depolymerization of microtubules, shown by immunofluorescence microscopy and measured as a 70.9 +/- 6.9% (n = 11, P<0.01) decrement by immunoblot analysis. These changes, which occur only when extracellular Pi concentrations are kept low, seem to be related to a significant increase within 5 min in the rate of cellular Pi uptake by 25.5% under these conditions. The shifts in the dynamic equilibria between monomeric and polymerized actin and tubulin in response to cellular Pi uptake were transient, being fully reversible within 30 min. Moreover, the effect of Pi seemed to be specific because inhibition of its uptake by phosphonoformic acid blunted microtubular disassembly markedly. In contrast, measurement of Pi uptake in the presence of agents known to stabilize cytoskeletal structures showed a substantial decrease with phallacidin, which stabilized microfilaments, whereas the microtubule stabilizer taxol had no apparent effect. These results indicate that acute alterations in the polymerization dynamics and stability of both microfilaments and microtubules are involved in the modulation of Na+/Pi co-transport and suggest important cytoskeletal participation in proximal tubular transport functions.

Actin Cytoskeleton↗
PubMed1995

Water channel expression in human ADPKD kidneys.

Cyst enlargement in autosomal dominant polycystic kidney disease (ADPKD) results in part from the transport of solute and fluid into the lumen of the cyst. In proximal tubules and thin descending limbs of normal kidneys, the high transepithelial water permeability of these segments is due to the presence of the water channel protein, aquaporin-CHIP (AQP-CHIP, i.e., AQP-1). The collecting ducts of normal kidneys express another member of this gene family, the aquaporin collecting duct (AQP-CD, i.e., AQP-2). The expression and distribution of these two members of the aquaporin gene family were examined in ADPKD and normal human kidneys. In both tissues, Western blotting with the anti-AQP-CHIP antibody revealed a major 28-kDa band. By immunofluorescence, AQP-CHIP was present in proximal tubules and thin descending limbs of Henle of both normal and ADPKD kidneys. In the latter, AQP-CHIP was detected in the epithelia lining 71% of cysts. Many cysts were positive for the proximal tubule marker gp330 (44%). Some cysts expressing AQP-CHIP did not stain for gp330, suggesting a descending thin limb origin, and a few cysts were negative for both markers. In normal human kidney, Western blotting with the anti-AQP-CD antibody revealed a band at 28 kDa. AQP-CD was localized to collecting ducts and did not show colocalization with gp330 in normal human kidney. In ADPKD kidney, AQP-CD was expressed by only 8% of cysts. In summary, water channels, primarily AQP-CHIP, are expressed in epithelial cells lining cysts in approximately 80% of cysts in ADPKD kidneys.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1986

Sites of insulin and glucagon metabolism in the rabbit nephron.

Uptake and degradation of peptide hormones have been demonstrated in proximal convoluted tubules (PCT), but the contribution of other regions of the nephron to renal hormone metabolism is unknown. In the present study we used micromethods to determine for the first time the degradation of radiolabeled insulin and glucagon by each segment of the rabbit nephron and examined some characteristics of this process in proximal convoluted and cortical collecting tubules (CCT). Degradation of insulin (8-10 fmol . cm-1 . h-1) and glucagon (13-15 fmol . cm-1 . h-1) was maximal in proximal convoluted and straight tubules, but occurred to a substantial degree (approximately 25-30% of PCT) in all other segments of the nephron except the thin descending limb. In PCT degradation of both hormones was maximal at physiological pH, and competition studies suggested that it is brought about by both specific and nonspecific proteases. Most of the degrading activity (insulin, 86%; glucagon, 73%) was in the cytosol or could be eluted off the cell membrane or organelles. In the CCT, a representative segment of the distal nephron, the characteristics of insulin degradation were similar to those observed in the PCT, whereas glucagon degradation appeared to be due chiefly to nonspecific proteases. In conclusion, the metabolism of insulin and glucagon by isolated rabbit tubules occurs chiefly in the proximal convoluted and straight tubules, but up to one-third of the degrading capacity of the proximal nephron is also present in distal nephron segments.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Marked proteinuria in hypertensive nephrosclerosis.

Heavy proteinuria in patients with essential hypertension raises the question of underlying primary renal disease. While malignant hypertension may be associated with proteinuria in the nephrotic range, it is generally held that protein excretion in benign nephrosclerosis is almost invariably less than 0.5-1.0 g/24 h. We report 18 patients with biopsy-proven hypertensive nephropathy and heavy proteinuria, of which only 6 had malignant hypertension. In the remaining 12 patients with benign nephrosclerosis, protein excretion reached up to 6.5 g/24 h, and nephrotic range proteinuria was present in 3 patients. All patients with heavy proteinuria suffered from long-standing moderate or severe, poorly controlled hypertension and were azotemic. We suggest that hypertensive nephrosclerosis be included in the differential diagnosis of massive proteinuria accompanying azotemia in poorly controlled hypertensives.

Adult↗
PubMed1985

First exchange neutrophilia: an index of peritonitis during chronic intermittent peritoneal dialysis.

During intermittent peritoneal dialysis (IPD) early diagnosis of peritonitis may be difficult, because of paucity in clinical signs and delays in bacteriologic studies. We examined prospectively leucocyte counts and their differential composition in initial ascites and dialysis effluent of patients on IPD and correlated these findings to the presence of subsequently bacteriologically proven clinical peritonitis. Total leucocyte counts from either ascites or first exchange effluent did not differentiate infected from noninfected patients. In contrast, first exchange effluent neutrophilia (greater than 43%) proved to be an early indicator of infection, being 100% sensitive and 94% specific for peritonitis. We conclude that in such patients peritoneal effluent neutrophilia should be considered an indication of possible infection.

Adult↗
PubMed1984

Role of the kidney in metabolism of gonadotropins in rats.

The role of the kidney in the metabolic disposal of homologous gonadotropins [renal luteinizing hormone (rLH) and renal follicle-stimulating hormone (rFSH)] was studied in rats. In analogy with other protein hormones, renal mechanisms contributed importantly to their metabolic clearance rates (MCR), which were profoundly and comparably decreased following nephrectomy (by 94 and 78% for rLH and rFSH, respectively). Absolute MCR and renal organ clearance rates of gonadotropins were, however, markedly lower and urinary clearance rates proportionally higher than those of nonglycosylated protein hormones reported previously. Nonetheless, handling of both LH and FSH by the kidney probably involves, in addition to their excretion in the urine, also intrarenal degradation because their urinary clearance rates accounted for at most a third of their respective MCR, considerably less than the striking reduction of MCR seen after acute renal ablation. Moreover, losses of LH immunoreactivity across the renal circulation were over and above those accountable for by urinary excretion alone. Thus, handling of gonadotropins by the kidney differs from that of nonglycoprotein hormones both in magnitude and in that it involves, in addition to intrarenal degradation, also substantial urinary excretion, a pattern that appears to be representative of the way the kidney disposes of glycoprotein hormones in general.

Animals↗
PubMed1984

Metabolism of pure human erythropoietin in the rat.

The metabolism of pure human erythropoietin (EPO) labeled with 125I was studied in the rat. Concentrations of the labeled hormone (125I-EPO) in plasma and urine were measured by both trichloroacetic acid precipitation and gel filtration. During steady-state conditions the metabolic clearance rate of 125I-EPO was slow, averaging 256 +/- 7 microliter. min-1 X kg-1 of which only 19 +/- 2 microliter X min-1 X kg-1 (or 7.4 +/- 0.8% of the metabolic clearance rate) could be accounted for by excretion of the labeled hormone in the urine. Urinary clearance of 125I-EPO amounted to less than 0.3% of the glomerular filtration rate, and there was no detectable arteriovenous concentration difference of 125I-EPO across the kidney. After both pulse injection and constant infusion to equilibrium, disappearance of 125I-EPO from the circulation could be approximated by a single exponential function: plasma half-life was 3.5 +/- 0.2 h in normal rats and was prolonged to 4.4 +/- 0.3 h (P less than 0.05) in animals with ligated renal pedicles. Although kidney homogenates degraded 125I-EPO in vitro (optimum pH 4.5), the hormone did not accumulate in the kidney when injected intravenously. We conclude that EPO metabolism is extremely sluggish compared with that of other polypeptide hormones. Whereas kidney tissue is capable of degrading EPO in vitro, the physicochemical characteristics of this glycoprotein (molecular size, shape, and charge) probably impede its access to degrading sites and therefore account for the limited contribution of renal extraction and excretion to the metabolic clearance of the hormone.

Animals↗
PubMed1983

Glucagon degradation by luminal and basolateral rabbit tubular membranes.

Glucagon is avidly degraded by the kidney, but the relative contribution of the luminal and basolateral tubular membranes to this process is unknown. We studied 125I-glucagon degradation by purified luminal (L) and basolateral (BL) tubular membranes prepared from rabbit kidney cortex, which showed enrichment vs. homogenate of marker enzyme activities (Na-K-ATPase for BL and maltase for L) of 10- and 14-fold, respectively. Renal homogenates and both tubular membrane fractions degraded glucagon avidly without reaching saturation even at pharmacologic concentration (10(-5) M) of the hormone. At physiologic concentration (3 x 10(-11) M) BL membranes degraded substantial amounts of glucagon (8.1 +/- 0.9 pg . micrograms protein-1 . h-1) even though at lesser rates (P less than 0.001) than the luminal fraction (33.3 +/- 1.9 pg . micrograms protein-1 . h-1). Competition experiments suggested that glucagon-degrading activity in both fractions includes both specific and nonspecific components, and the potency of different enzyme inhibitors to decelerate glucagon degradation was strikingly similar in the two membrane preparations. Glucagon degradation differed in several important aspects from the manner in which tubular membranes catabolize insulin, including absolute degradation rates and relative degrading capacity of the membranes vs. homogenates, both being substantially higher for glucagon. These results provide direct evidence that the renal metabolism of glucagon also involves its degradation by peritubular cell membranes.

Animals↗
PubMed1983

Insulin binding sites in various segments of the rabbit nephron.

Insulin binds specifically to basolateral renal cortical membranes and modifies tubular electrolyte transport, but the target sites of this hormone in the nephron have not been identified. Using a microassay that permits measurement of hormone binding in discrete tubule segments we have determined the binding sites of 125I-insulin along the rabbit nephron. Assays were performed under conditions that minimize insulin degradation, and specific binding was measured as the difference between 125I-insulin bound in the presence or absence of excess (10(-5) M) unlabeled hormone. Insulin monoiodinated in position A14 was used in all assays. Specific insulin binding (attomol . cm-1 +/- SE) was highest in the distal convoluted tubule (180.5 +/- 15.0) and medullary thick ascending limb of Henle's loop (132.9 +/- 14.6), followed by the proximal convoluted and straight tubule. When expressed per milligram protein, insulin binding capacity was highest along the entire thick ascending limb (medullary and cortical portions) and the distal convoluted tubule, i.e., the "diluting segment" (congruent to 10(-13) mol . mg protein-1), and was lower (congruent to 4 X 10(-14) mol . mg protein-1), and remarkably similar, in all other nephron segments. Binding specificity was verified in competition studies with unlabeled insulin, insulin analogues (proinsulin and desoctapeptide insulin), and unrelated hormones (glucagon, 1-34 parathyroid hormone, prolactin, follicle-stimulating hormone). In addition, serum containing antiinsulin receptor antibody from two patients with type B insulin resistance syndrome markedly inhibited insulin binding to isolated tubules. Whether calculated per unit tubule length or protein content, insulin binding is highest in the thick ascending limb and the distal convoluted tubule, the same nephron sites where a regulatory role in sodium transport has been postulated for this hormone.

Animals↗
PubMed1982

Insulin binding and degradation by luminal and basolateral tubular membranes from rabbit kidney.

Insulin influences certain metabolic and transport renal functions and is avidly degraded by the kidney, but the relative contribution of the luminal and basolateral tubular membranes to these events remains controversial. We studied (125)I-insulin degradation [TCA and immunoprecipitation (IP) methods] and the specific binding of the hormone by purified luminal (L) and basolateral (BL) tubular membranes. These were prepared from rabbit kidney cortical homogenates by differential and gradient centrifugation and ionic precipitation steps in sequence, which resulted in enrichment vs. homogenate of marker enzymes' activities (sodium-potassium-activated adenosine triphosphatase for BL and maltase for L) of 8- and 12-fold, respectively. Both fractions degraded insulin avidly and bound the hormone specifically without saturation even at pharmacologic concentrations (10 muM). At physiologic insulin concentrations (0.157 nM) BL membranes degraded substantial amounts of insulin (44.2+/-2.6 and 40.7+/-2.2 pg/mg protein per min by the TCA and IP methods, respectively), even though at lesser rates (P < 0.001) than the luminal fraction (67.2+/-2.3 and 75+/-6.2 pg/mg protein per min, respectively); the rate of insulin catabolism by BL membranes was significantly higher (P < 0.001) than that which could be attributed to their contamination by luminal components [12.2+/-1.9 pg/mg per min (TCA method), or 13.7+/-1.9 pg/mg per min (IP method)]. Competition experiments suggested that insulin-degrading activity in both fractions includes both specific and nonspecific components. In contrast to degradation, insulin binding by both membranes was highly specific for native insulin and was severalfold higher in BL than L membranes [17.5+/-1.3 vs. 4.5+/-0.4 fmol/mg protein (P < 0.001) at physiologic insulin concentrations]. Despite the marked difference in the binding capacity for insulin by the two membranes, the patterns of labeled insulin displacement by increasing amounts of unlabeled hormone were superimposable (50% displacement required approximately 3 nM), suggesting that their receptors' affinity for insulin was similar. These observations provide direct evidence that interaction of insulin with the kidney involves binding and degradation of the hormone at the peritubular cell membrane.

Animals↗
PubMed1981

Prolactin metabolism in the rat: role of the kidney in degradation of the hormone.

The contribution of impaired prolactin (PRL) degradation to the altered dynamics of this hormone in uremia was investigated in rats. Hyperprolactinemia developed after bilateral nephrectomy (BNx) or ligation of both ureters (BUL), whereas PRL levels remained normal in comparably azotemic animals undergoing urine autoinfusion in which glomerular filtration rate (GFR) was maintained. The renal organ clearance of PRL in control rats accounted for two-thirds of its metabolic clearance rate and was consistently less than GFR. Following BUL and BNx the metabolic clearance of PRL decreased predictably also by two-thirds. The importance of the renal parenchyma in the degradation of prolactin was confirmed during perfusion of isolated rat kidneys. Renal PRL handling involves mainly glomerular filtration and tubular reabsorption, although uptake from peritubular blood is also demonstrable under the high plasma flow conditions obtaining during in vitro kidney perfusion. We conclude that the hyperprolactinemia associated with acute uremia in the rat is not the consequence of the uremic state per se, but results from impaired renal degradation of the hormone.

Animals↗