Isolation and identification of two cyclosporine aldehydic metabolites from the bile of a liver transplant recipient during an acute nephrotoxic episode.
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Animals↗
Biomedical subjects
K Skorecki
Publications and source records attributed to K Skorecki.
Refractory ascites in cirrhosis: roles of volume expansion and plasma atrial natriuretic factor level elevation.
Cirrhotic patients with ascites refractory to diuretics also have blunted response to marked elevations of plasma atrial natriuretic factor levels alone or to moderate intravascular volume expansion by head-out water immersion. However, these patients usually undergo natriuresis after peritoneovenous shunting. To dissect the factors responsible for this response, we studied the effects on separate days of moderate intravascular volume expansion and highly elevated plasma atrial natriuretic factor levels (head-out water immersion and atrial natriuretic factor infusion) or marked volume expansion and moderate plasma atrial natriuretic factor level elevation (head-out water immersion and albumin infusion) in 13 alcoholic cirrhotic patients with massive ascites. Three of these patients, who responded to initial head-out water immersion with a negative sodium balance, served as controls. Unresponsiveness to head-out water immersion was confirmed in the remaining 10 patients on both days on the basis of blunted natriuretic response (urinary sodium excretion < 0.8 mmol/hr after 2 hr). In contrast, these 10 refractory patients were able to achieve negative sodium balance with both combinations. Mean urinary sodium excretion increased from a baseline level of 0.13 +/- 0.10 mmol/hr to a peak level of 2.29 +/- 0.61 mmol/hr after head-out water immersion and atrial natriuretic factor infusion and from 0.10 +/- 0.3 mmol/hr to 1.61 +/- 0.62 mmol/hr after head-out water immersion and albumin infusion. Both maneuvers were associated with suppression of plasma renin activity and serum aldosterone levels. With head-out water immersion and atrial natriuretic factor infusion, we noted a significant increase in 5' cyclic GMP levels, a second messenger of atrial natriuretic factor, indicating possible activation of atrial natriuretic factor receptors at the inner medullary collecting ducts. In contrast, with head-out water immersion and albumin infusion no such increase in levels occurred, indicating that the increase in urinary sodium excretion was mainly due to increased delivery of sodium to the cortical distal nephron, as indicated by a disproportionate increase in urinary potassium excretion. In conclusion, massive (as opposed to moderate) volume expansion or greatly elevated levels of plasma atrial natriuretic factor associated with moderate volume expansion can improve blunted atrial natriuretic factor responsiveness in cirrhotic patients with refractory ascites. This appears to be achieved by way of a marked increase in distal delivery of filtrate in the kidney, with or without activation of distal atrial natriuretic factor receptors in the inner medullary collecting ducts.
Adult↗
Atrial natriuretic factor and liver disease.
A working formulation for the role of ANF in the sodium retention of cirrhosis is summarized in Figure 4. Sodium retention is initiated early in cirrhosis, either as a result of hepatic venous outflow block or of primary vasodilation. The consequent intravascular volume expansion causes increases in ANF levels. At this stage of disease, the rise in ANF level is sufficient to counterbalance the antinatriuretic influences. However, this occurs at the expense of an expanded intravascular volume with the potential for overflow ascites. With progression of disease, disruption of intrasinusoidal Starling forces and loss of volume from the vascular compartment into the peritoneal compartment occur. This underfilling of the circulation may attenuate further increases in plasma ANF and promotes the activation of antinatriuretic factors. At this later stage of disease, elevated levels of ANF are insufficient to counterbalance antinatriuretic influences. Thus the role of ANF in cirrhosis is primarily beneficial in that it successfully attenuates the antinatriuretic forces in the compensated stage. Raised ANF levels have two potential deleterious effects. First, ANF may exacerbate arterial vasodilation, leading to further sodium retention. The primacy of vasodilatation has been proposed as an alternate formulation to the overflow and underfill hypotheses. Second, Epstein et al. found higher basal ANF levels in cirrhotic patients with edema than in those patients without edema. ANF is known to reduce plasma volume in anephric animals and to increase the ultrafiltration coefficients of isolated capillaries. Therefore it is conceivable that in the clinical setting in which antinatriuretic factors limit the renal responsiveness to ANF but in which ANF levels are elevated (i.e., cirrhosis, congestive heart failure, primary kidney disease), ANF itself may contribute to edema formation at the level of the peripheral microcirculation. In general, ANF likely has no primary role in the sodium retention in cirrhosis. In early compensated cirrhosis, ANF may maintain sodium homeostasis despite the presence of mild antinatriuretic factors. In late ascitic cirrhosis renal resistance to ANF develops, rendering it ineffective.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Bead transfection: rapid and efficient gene transfer into marrow stromal and other adherent mammalian cells.
We report a simple, rapid, efficient and cost-effective method of gene transfer into bone marrow stromal and other adherent mammalian cells. Our approach involves brief incubation of cells with glass beads in a solution containing the DNA to be transferred. We optimized the technique using COS cells (SV40 transformed kidney cell line from African green monkey) and a transient expression assay for chloramphenicol acetyl transferase (CAT). Factors affecting gene transfer include size and condition of the beads and DNA concentration, but not DNA conformation. Gene transfer efficiency, assessed in a transient expression assay for beta-galactosidase activity, was 5 and 3% in nontransformed human bone marrow stromal cells and COS cells, respectively. Long-term stable expression with the selectable marker, neomycin phosphotransferase, was demonstrated in clonogenic COS cells at a frequency of 27%. Southern analysis of resistant clones revealed the transferred DNA to be integrated in low copy number at one or two sites in the host cell genome. Comparison with electroporation and DEAE-dextran indicates that bead transfection is more efficient than the latter and less costly than either of these methods. In view of its simplicity and because the use of retroviral sequences can be avoided, bead transfection may be an attractive means of gene insertion for gene therapy.
Animals↗
Promoter region of the murine fibroblast growth factor receptor 2 (bek/KGFR) gene.
Fibroblast growth factors (FGFs) and their receptors play an important role in cell growth, angiogenesis and embryonal development. Four distinct genes encoding fibroblast growth factor receptors (FGFRs) were identified: flg, encoding FGFR1, bek encoding FGFR2, and the genes for FGFR3 and FGFR4. Both FGFR2 and keratinocyte growth factor receptor (KGFR) are encoded by the same gene, bek. To study the regulation of expression of the FGF receptors we analysed the DNA sequence flanking the 5' region of the cDNA of murine FGFR2 to seek elements that control its transcription. A 5-kbp fragment containing the 5' end of the cDNA was isolated from mouse genomic library and used to map the promoter region. We found that the sequence encoding the 5' non-translated region of the FGFR2/KGFR cDNA contains an intron located 210 bp upstream from the translation start site. Using RNAase protection and primer extension, we identified the mRNA start 37 bp upstream from the beginning of the bek cDNA. The promoter activity was found to reside in a 1.3-kbp fragment upstream from the cDNA, and deletion mapping further localized the promoter to a 0.7-kbp fragment. The sequence of this region shows high G+C content (62%), which is particularly emphasized in the 200 bp upstream from the mRNA start (80% G+C). This region contains the CCGCCC, GGGCGG AND GGAGG motifs also found in promoters of other growth factor receptors. Neither TATA nor CAAT boxes were found near the RNA start site. The characterization of this promoter will allow studies of the regulation of expression of the FGFR2 during development and in pathophysiological states. The differences between the promoter sequence of the gene for FGFR2 (bek) and FGFR1 (flg) may explain their differential expression during development.
Animals↗
Carboxy-terminal truncations of epidermal growth factor (EGF) receptor affect diverse EGF-induced cellular responses.
The binding of epidermal growth factor (EGF) to its receptor induces tyrosine phosphorylation of phospholipase C gamma (PLC gamma), which appears to be necessary for its activation leading to phosphatidyl inositol (PI) hydrolysis. Moreover, EGF-receptor (EGF-R) activation and autophosphorylation results in binding of PLC gamma to the tyrosine phosphorylated carboxy-terminus of the receptor. To gain further insights into the mechanisms and interactions regulating these processes, we have analyzed transfected NIH-3T3 cells expressing two EGF-R carboxy-terminal deletion mutants (CD63 and CD126) with reduced capacity to stimulate PI hydrolysis, Ca2+ rises, and DNA synthesis. In fact, the CD126 mutant lacking 126 carboxy-terminal amino acids, including four tyrosine autophosphorylation sites, was unable to stimulate PI hydrolysis or Ca2+ rise in response to EGF. Surprisingly, EGF binding to the cell lines expressing CD63 or CD126 mutants was followed by similar stimulation of tyrosine phosphorylation of PLC gamma. Our results suggest that although necessary, tyrosine phosphorylation of PLC gamma may not be sufficient for stimulation and PI hydrolysis. It is clear, however, that the carboxy-terminal region of EGF-R is involved in regulation of interactions with cellular targets and therefore plays a crucial role in postreceptor signaling pathways.
3T3 Cells↗
Effect of phospholipase C-gamma overexpression on PDGF-induced second messengers and mitogenesis.
Platelet-derived growth factor (PDGF) stimulates phospholipase C (PLC) activity and the phosphorylation of the gamma isozyme of PLC (PLC-gamma) in vitro and in living cells. The role of PLC-gamma in the phosphoinositide signaling pathway was addressed by examining the effect of overexpression of PLC-gamma on cellular responses to PDGF. Overexpression of PLC-gamma correlated with PDGF-induced tyrosine phosphorylation of PLC-gamma and with PDGF-induced breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2). However, neither bradykinin- nor lysophosphatidic acid-induced phosphoinositide metabolism was enhanced in the transfected cells, suggesting that the G protein-coupled phosphoinositide responses to these ligands are mediated by other PLC isozymes. The enhanced PDGF-induced generation of inositol trisphosphate (IP3) did not enhance intracellular calcium signaling or influence PDGF-induced DNA synthesis. Thus, enzymes other than PLC-gamma may limit PDGF-induced calcium signaling and DNA synthesis. Alternatively, PDGF-induced calcium signaling and DNA synthesis may use biochemical pathways other than phosphoinositide metabolism for signal transduction.
Animals↗
A role for G-proteins in the epidermal growth factor stimulation of phospholipase A2 in rat kidney mesangial cells.
We have previously demonstrated phospholipase C (PLC) independent activation of phospholipase A2(PLA2) by epidermal growth factor (EGF) in glomerular mesangial cells in culture. In the current study using glass beads to permeabilize [3H]- or [14C]-arachidonate labelled mesangial cells we demonstrate that guanine nucleotides modulate the EGF-mediated stimulation of arachidonic acid release (75% inhibition with 100 microM GDP beta S and 108% augmentation with 100 microM GTP gamma S). GTP gamma S alone stimulated both the release of free arachidonic acid and production of diacylglycerol (DAG), while EGF itself neither stimulated DAG nor augmented the DAG response to GTP gamma S. These findings suggest the intermediacy of a G-protein in PLC-independent stimulation of PLA2 by a growth factor, and provide a model system for determining the relationship between G-protein intermediacy and the intrinsic tyrosine kinase activity of the growth factor receptor.
Animals↗
The cellular action of antidiuretic hormone.
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Adenylyl Cyclases↗
The effects of cyclosporine and cyclosporine metabolites in experimental small intestinal transplantation.
Cyclosporine metabolites (CM) were compared with cyclosporine for their in vitro and in vivo immunosuppressive, nephrotoxic, and hepatotoxic effects using (A) in vitro mixed lymphocyte induction of monocyte/macrophage procoagulant activity (PCA), an accurate marker of allograft rejection; (B) in vitro toxic effects on renal cells in culture; and (C) a unidirectional rejection model of rat small intestinal transplantation (SIT). CM were composed of OL1, OL17, OL18, and two additional peaks C and H, (peak C: mass = 1235, 15.3% of total CM, peak H: mass = 1205, 6.3% of total CM). In vitro, CM fully suppressed the one-way mixed lymphocyte culture-induced PCA from 52.5 +/- 8.2 mU/10(6) PBM to the basal level 22.3 +/- 6.6 mU/10(6) PBM (P less than 0.01), which was comparable to CsA (21.3 +/- 5.5 mU/10(6) PBM). Lewis rats that had received Lewis-Brown Norway F1 hybrid intestinal allografts when treated with CM, demonstrated near-normal histology with minimal signs of rejection as compared with the fulminant clinical and histological rejection observed in the control (untreated and Cremaphor/NaCl treated) animals. PCA was markedly elevated in the control animals, 278 +/- 172 (untreated) and 160 +/- 98 mU/10(6) PBM (Cremaphor/normal saline treated), whereas CsA-treated allogeneic transplants expressed only basal levels of PCA (14.0 +/- 4 mU/10(6) PBM) (P less than 0.01), associated with normal histology. CM-treated animals expressed PCA levels of 27.0 +/- 10 mU/10(6) PBM, which was significantly different from both control and CsA-treated animals (P less than 0.01). In contrast to CsA-treated animals, CM-treated allogeneic transplants demonstrated no apparent renal or hepatic toxicity, as measured by blood urea nitrogen (25.3 +/- 9.5 vs. 10.0 +/- 5.3 mg/dl), alkaline phosphatase (160.7 +/- 29.3 vs. 100.3 +/- 19.5 U/L), and aspartate transaminase (96.7 +/- 23.7 vs. 61.7 +/- 11.7 U/L) (P less than 0.01). Similarly, in contrast to CsA, CM had minimal or no toxicity in renal epithelial and mesangial cells in culture, as measured by minimal or no inhibition of DNA, RNA, and protein synthesis. These results suggest that CM have potent immunosuppressive properties with no apparent nephrotoxicity and hepatotoxicity in vitro and in vivo.
Animals↗
A novel calcium signalling response in the breast cancer cell line MDA-468.
In the human breast carcinoma cell line MDA-468 addition of epidermal growth factor (EGF) is growth inhibitory. Calcium signalling was investigated in this cell line using the calcium sensitive fluorescent probe Indo-1. Addition of EGF to MDA-468 cells resulted in a novel biphasic calcium response. In the first phase of the response EGF raised calcium to levels significantly above basal. This was followed by a prolonged fall in calcium to levels significantly lower than original basal levels. The G-protein activator aluminum fluoride (AlF), stimulated a rise in calcium which was not proceeded by a fall below basal levels. Conversely addition of PMA, an activator of protein kinase C (PKC), induced a fall in calcium from basal without a prior increase. Down regulation of PKC eliminated the response to PMA, however the biphasic nature of the EGF response was maintained. Pretreatment of the cells with pertussis toxin did not alter the response to EGF nor to AlF. We conclude that in the MDA-468 cell in which EGF is growth inhibitory: 1) EGF results in a biphasic calcium response which ultimately leads to reduction below baseline levels, 2) a rise in calcium itself is not sufficient to account for the subsequent fall below basal levels, 3) G-proteins may be involved in the initial phase of the EGF response, 4) activation of PKC can also reduce intracellular calcium, however the response to EGF is not dependent on this pathway.
Aluminum↗
Toxic effects on renal cells in culture--a comparison of cyclosporin A and its metabolites.
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Animals↗
Cyclosporine induced alterations in vasopressin signalling in the glomerular mesangial cell.
Nephrotoxicity is the major limiting factor in the clinical use of cyclosporine A (CyA), which in its acute form results in a decrease in renal blood flow and glomerular filtration rate. Contractile mesangial cells regulate glomerular capillary patency in response to vasoactive stimuli including vasopressin. Hence the effects of CyA on the cellular signalling mechanism of vasopressin in cultured glomerular mesangial cells were investigated. Vasopressin induced a transient rise in intracellular calcium which was enhanced in the presence of CyA. In addition, CyA decreased both basal and vasopressin stimulated PGE2 production. This inhibition was mediated at least in part at the level of arachidonate release and may involve inhibition of phospholipase A2. We conclude that the increased renal and systemic vasoconstriction that occurs with CyA treatment may be the result of an exaggerated rise in intracellular calcium combined with diminished production of vasorelaxant prostaglandins in response to vasoconstrictor hormones.
Animals↗
Reduced cyclosporin accumulation in multidrug-resistant cells.
Cyclosporin accumulation was reduced by 50% or more in multidrug- resistant CHRC5 CHO cells with high levels of P-glycoprotein expression compared to drug sensitive AuxB1 CHO cells. This difference could be overcome by verapamil which is known to interact with P-glycoprotein and reverse multidrug resistance. The difference in cyclosporin accumulation between sensitive and resistant cells decreased with increasing cyclosporin concentrations suggesting that cyclosporine itself regulated its own accumulation through interaction with P-glycoprotein. Indeed, cyclosporin also reversed differences in vinblastine accumulation between resistant and sensitive cell lines. Since P-glycoprotein is highly expressed in the kidney which is also a target for cyclosporin toxicity, the effects of verapamil on cyclosporin accumulation were studied in two renal cell lines, rat mesangial cells and LLCPK1, cells. Verapamil increased cyclosporin accumulation by approximately 70%. These results suggest that cellular cyclosporine accumulation is regulated at least in part by its interaction with P-glycoprotein.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Interaction of atrial natriuretic peptide-stimulated guanylate cyclase and vasopressin-stimulated calcium signaling pathways in the glomerular mesangial cell.
Receptors for atrial natriuretic peptide (ANP) have been demonstrated in renal mesangial cells as well as other cell types in the glomerulus. The biochemical basis for the effects of ANP on glomerular hemodynamics remains undefined. Using cultured rat glomerular mesangial cells, we demonstrated a concentration-dependent stimulation of cGMP production in intact cells, and of guanylate cyclase in membranes. Despite the presence of a guanylate cyclase response, ANP had no inhibitory effect on basal inositol trisphosphate production nor on basal cytosolic calcium. Arginine vasopressin stimulated IP3 production, caused a rise in cytosolic calcium as measured using the calcium-sensitive fluorescent probe Indo-1, and caused mesangial cell contraction. ANP caused a slight but significant enhancement of vasopressin-stimulated IP3 production, but had no effect on the cytosolic calcium response nor on the contractile response. 8-Bromo-cGMP likewise had no effect on the generation of the calcium signal. These results indicate that the effects of ANP on glomerular hemodynamics are not mediated by an alteration in the generation of the calcium signal in mesangial cells. In contrast, addition of calcium inhibited ANP stimulated guanylate cyclase activity.
1-Methyl-3-isobutylxanthine↗
Bradykinin stimulates a rise in cytosolic calcium in renal glomerular mesangial cells via a pertussis toxin insensitive pathway.
Bradykinin elicits a complex response in the renal glomerulus which includes a reduction in the glomerular capillary ultrafiltration coefficient. To elucidate the biochemical mechanism of this response, we investigated calcium signalling in rat renal glomerular mesangial cells in culture using the calcium-sensitive fluorescent dye, Indo-1. Bradykinin was found to cause a concentration-dependent transient rise in cytosolic free calcium followed by a sustained slower secondary rise. The bradykinin response persisted with acute removal of extracellular calcium using EGTA, indicating that calcium entry from outside the cell did not mediate this primary response. Prolonged exposure to EGTA, which reduced intracellular stores, eliminated the calcium response to bradykinin but not to vasopressin, indicating differential sensitivity to intracellular calcium stores of these two hormonal responses. In agreement, prior stimulation with vasopressin significantly attenuated the response to bradykinin, but the converse did not occur. Aluminum fluoride and pertussis toxin were used to investigate the possible involvement of a guanyl nucleotide regulatory protein in signal transduction. Aluminum fluoride induced a transient rise in cytosolic calcium that was abrogated by prior exposure of the cells to pertussis toxin. This demonstrates the effectiveness of pertussis toxin and the presence of a calcium-signalling pathway susceptible to pertussis toxin in these cells. In contrast, the responses to bradykinin and vasopressin were unaffected by pertussis toxin. We conclude that bradykinin stimulates release of calcium from intracellular stores in glomerular mesangial cells via a pertussis toxin insensitive pathway. This mesangial response provides a direct biochemical basis for the bradykinin-induced fall in glomerular capillary ultrafiltration coefficient which has been observed in vivo.
Animals↗
Mechanism of Li inhibition of vasopressin-sensitive adenylate cyclase in cultured renal epithelial cells.
We investigated the mechanism for lithium-induced inhibition of vasopressin-stimulated adensoine 3',5'-cyclic monophosphate (cAMP) production in the renal epithelial cell line LLC-PK1. In LLC-PK1 membranes lithium caused direct inhibition of hormone-stimulated adenylate cyclase activity by competing with magnesium. Fifty percent inhibition occurred at 20 mM lithium. The maximum transport activity (Vmax) but not the activation constant (Ka) for activation by vasopressin was altered. Activation by GTP and its nonhydrolyzable analogues was also inhibited by lithium. Furthermore, kinetic studies revealed that the lag phase in the activation of adenylate cyclase by 5'-guanylimi-dotriphosphate [Gpp(NH)p] was prolonged from 1 to 3 min, suggesting an effect of lithium on magnesium-dependent activation of the stimulatory GTP binding protein Gs. The function of the corresponding inhibitory GTP-binding protein Gi, as assessed by GTP inhibition of vasopressin-stimulated adenylate cyclase activity in the presence and absence of pertussis toxin pretreatment, was unaffected. Intact LLC-PK1 cells incubated in 10 mM lithium (approximate urinary concentration in lithium-treated patients) attained an intracellular lithium concentration of 17 mM, which led to a 40% reduction in cAMP formation. Magnesium loading of intact cells with the ionophore A23187 reversed the inhibitory effect of lithium. It is concluded that lithium directly inhibits the activation of vasopressin-sensitive adenylate cyclase in renal epithelia by competing with magnesium for activation of Gs. This direct effect on Gs activation accounts for the inhibitory effect of lithium on cAMP production in the intact cell.
1-Methyl-3-isobutylxanthine↗
Cyclosporine A in contrast to a cyclosporine metabolite (OL-17) specifically inhibits growth of renal cells in culture.
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Animals↗