Thrombin increases clusterin mRNA in glomerular epithelial and mesangial cells

Clusterin, a multifunctional protein with complement blocking activity, and fibrin, a product of thrombin's enzymatic activity, are present in the kidney during acute and chronic renal failure. The role of thrombin in regulating clusterin mRNA in the kidney is not known. The effect of thrombin on clusterin mRNA expression was examined in rat glomerular mesangial and glomerular epithelial cells, and cultured human renal proximal tubular epithelial cells by northern blot. Thrombin (10(-8) M) increased clusterin mRNA levels two- to fourfold in glomerular mesangial, glomerular epithelial, and proximal tubule epithelial cells. This was a specific effect of thrombin receptor activation because peptides corresponding to the tethered ligand of the thrombin receptor were also able to increase clusterin mRNA levels. Epidermal growth factor, insulin-like growth factor-1, and transforming growth factor-beta 1 had little or no effect on clusterin mRNA levels. The protein kinase C inhibitor RO-32-0432 (1 microM) inhibited the thrombin-induced increase in clusterin mRNA, suggesting that thrombin receptor activation may regulate renal clusterin mRNA levels through protein kinase C.     

Characterization of glomerular thromboxane receptors in murine lupus nephritis

Renal thromboxane (Tx) production is increased in the MRL-lpr murine model of lupus nephritis. To investigate the relationship between increased Tx production and number and affinity of Tx receptors, we measured binding of the Tx receptor antagonist [3H][SQ295481S-1 alpha,2 beta(5Z),3 beta,4 alpha]-7-(3-((2-((phenyl- amino)-carbonyl)hydrozino)methyl)-7-oxabicyclo-(2.2.1)heptan -2-yl)-5-heptenoic acid in glomerular preparations from MRL-lpr mice and both MRL(-)+/+ and LG/J controls. Renal Tx binding was first characterized in normal LG/J mice. In these animals, glomerular binding was specific, saturable and reversible. Scatchard analysis revealed a single class of high-affinity binding sites. We next evaluated Tx production and binding in 12- and 16-week-old MRL-lpr mice and MRL(-)+/+ controls. To assess renal Tx production, excretion of TxB2 was measured in urine. Urinary TxB2 was increased in MRL-lpr mice at 16 weeks of age. This increase in urinary TxB2 was associated with a reduction in density of glomerular Tx binding sites compared to either 12-week-old MRL-lpr mice or MRL(-)+/+ controls. Ligand binding affinity was similar in all groups. To investigate if this alteration in binding was specific for Tx, glomerular binding of [3H]angiotensin II was measured. In MRL-lpr mice, the number and affinity of glomerular angiotensin binding sites were similar at 12 and 16 weeks of age. Thus, in this murine model of lupus nephritis, enhanced renal Tx production is temporally associated with a decrease in glomerular Tx binding sites without a change in receptor affinity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transient up-regulation of ciliary neurotrophic factor receptor-alpha mRNA in axotomized rat septal neurons

PURPOSE: To determine prospectively the feasibility, complications, and mid- and long-term advantages of peripheral insertion of central catheters in infants and children. MATERIALS AND METHODS: During a 15-month period between March 1995 and June 1996, a total of 285 catheter placement attempts were made to peripherally insert central catheters in 183 pediatric patients (89 boys, 94 girls). Phlebographic guidance was used, and the catheters were inserted below the elbow in 99% of cases. Catheter insertion was indicated for prolonged antibiotic therapy in 108 patients (158 catheter placement attempts), hematologic or oncologic care in 24 patients (40 attempts), total parenteral nutrition in 16 patients (46 attempts), and venous access for fluid or blood in 35 patients (41 attempts). The success rate and complications were recorded along with the indication, patient age, and duration of catheter placement. RESULTS: One hundred fifty-two of 158 (96%) catheter placement attempts were successful in outpatients (n = 108), 124 of 127 (98%) in hospitalized patients (n = 75), and 70 of 73 (96%) in patients aged less than 1 year. Infection and pericatheter venous thrombosis were the main complications and were seen in 17 of 276 (6%) and one of 276 (0.3%) catheter placement attempts, respectively. Catheter occlusion occurred in 23 of 276 (8%) catheter placement attempts. CONCLUSION: Peripheral insertion of central catheters was highly feasible in infants and children with this protocol. Such catheters were well tolerated in the pediatric population with a low frequency of complications.     

Protease nexin 1 in the murine kidney: glomerular localization and up-regulation in glomerulopathies

Protease nexin 1 (PN-1), a potent serpin-class antiprotease, is thought to be synthesized in the murine kidney. However, neither the cellular localization of PN-1 synthesis nor its role has yet been defined. To address these questions, we determined by in situ hybridizations RNase protection assay and immunoblotting, the sites of PN-1 mRNA accumulation in normal mouse kidneys and the modulation of PN-1 expression in several pathological conditions. In normal kidneys, PN-1 mRNA was detected primarily in glomeruli, most likely in mesangial cells. The glomerular expression of PN-1 was substantially enhanced not only in lupus-like glomerulonephritis (induced by IgG3 monoclonal rheumatoid factors or occurring spontaneously in lupus-prone mice), but also in mild glomerular lesions associated with intracapillary thrombi induced by IgG3 anti-trinitrophenyl monoclonal antibodies. In contrast, no modulation of PN-1 mRNA levels was observed during the course of lipopolysaccharide-induced acute tubular necrosis. A constitutive PN-1 gene expression and its up-regulation during glomerular injury suggest a possible role for PN-1 in glomerular biology. In view of its high inhibitory activity towards thrombin, mesangial PN-1 may be involved in the control of glomerular coagulation following initial glomerular injuries.     

Expression of Hex mRNA in early murine postimplantation embryo development

Photographs frequently are used to document change in the management of hypertrophic scars. The purpose of this study was to design a scale for the analysis of photographs of hypertrophic scars and to test its reliability. The subjects were four occupational and physical therapists, (two novices and two experts), in scar management. Existing scales were modified to produce a new scale. The subjects twice rated four slides from each of ten patients' scars, in random order. They used a Latin Square design. Interrater and test-retest reliabilities were calculated using a weighted kappa statistic. The newly developed scale demonstrated interrater reliability, which ranged between 0.66 and 0.90 for all items. The test-retest reliability ranged between 0.73 and 0.89 for all items. The new scale had substantial reliability (using a single rater) and was at least as reliable when used by novice therapists. This indicated that training had no effect.     

Activation of protein kinase A prevents the ethanol-induced up-regulation of delta-opioid receptor mRNA in NG108-15 cells

We have used a sensitive solution hybridization assay with a riboprobe transcribed from the coding sequence of the delta-opioid receptor gene (DOR) to study the up-regulation of the DOR mRNA by ethanol in NG108-15 cells. Exposure of the cells to compounds that increase cAMP levels (forskolin, forskolin + IBMX, or dibutyryl cAMP) resulted in the attenuation of ethanol-induced up-regulation of DOR mRNA. The inactive analogue of forskolin, 1,9-dideoxy forskolin had no effect. Northern blot analysis of RNA extracts from ethanol-, forskolin- or ethanol + forskolin-treated cells showed proportional changes in each of the multiple DOR mRNA bands, so that no difference was observed in the fraction of the total hybridization signal produced by each band of the DOR mRNA. In the absence of ethanol, forskolin or dibutyryl cAMP reduced the basal levels of DOR mRNA. The cAMP analogue (Rp)-cAMPS, a protein kinase A (PKA) inhibitor, increased DOR mRNA levels. However, the combination of (Rp)-cAMPS and ethanol did not further increase DOR mRNA levels compared to ethanol or (Rp)-cAMPS alone. Signaling through cAMP and PKA down-regulates DOR mRNA levels. The ethanol-induced increase in DOR mRNA levels in NG108-15 cells appears to be mediated via a reduction of PKA.     

Involvement of IL-16 in the induction of airway hyper-responsiveness and up-regulation of IgE in a murine model of allergic asthma

Experiments were designed to investigate the role of IL-16 in a mouse model of allergic asthma. OVA-sensitized mice were repeatedly exposed to OVA or saline aerosols. Bronchoalveolar lavage fluid (BALF) was collected after the last aerosol, and the presence of IL-16 was evaluated using a migration assay with human lymphocytes. Migration of lymphocytes was significantly increased in the presence of cell-free BALF from OVA-challenged mice compared with BALF from saline-challenged controls. This response was significantly inhibited after addition of antibodies to IL-16, demonstrating the presence of IL-16 in BALF of OVA-challenged animals. Immunohistochemistry was performed and revealed IL-16 immunoreactivity particularly in airway epithelial cells but also in cellular infiltrates in OVA-challenged mice. IL-16 immunoreactivity was absent in nonsensitized animals; however, some reactivity was detected in epithelial cells of sensitized but saline-challenged mice, suggesting that sensitization induced IL-16 expression in airway epithelium. Treatment of mice with antibodies to IL-16 during the challenge period significantly suppressed up-regulation of OVA-specific IgE in OVA-challenged animals. Furthermore, antibodies to IL-16 significantly inhibited the development of airway hyper-responsiveness after repeated OVA inhalations, whereas the number of eosinophils in bronchoalveolar lavage or airway tissue was not affected. In conclusion, IL-16 immunoreactivity is present in the airways after sensitization. After repeated OVA inhalation, IL-16 immunoreactivity is markedly increased and IL-16 is detectable in BALF. Furthermore, IL-16 plays an important role in airway hyper-responsiveness and up-regulation of IgE but is not important for eosinophil accumulation in a mouse model of allergic asthma.     

Characterization of the heparin-binding properties of human clusterin

Clusterin is a highly conserved mammalian glycoprotein which has been predicted to contain heparin-binding sites. We tested this prediction by studying the interactions between heparin and clusterin using ELISA and heparin affinity chromatography methodologies. Two forms of biotinylated heparin were used in ELISA: heparin which had been directly biotinylated with a biotin-N-hydroxysuccinimide ester and heparin which had been activated using epichlorohydrin and 1,6-diaminohexane prior to biotinylation. Both gave dose-dependent increases in ELISA signal with increasing concentrations of biotinylated heparin, with the latter giving signals an order of magnitude greater than the former. There was a dose-dependent increase in the ELISA signal from bound biotinylated heparin with increasing concentrations of plate-bound clusterin. The apparent affinity constant for binding of biotinylated heparin to plate-bound clusterin at pH 6.0 was estimated as 0.06 +/- 0.02 microM. Unlabeled heparin blocked the binding of biotinylated heparin to clusterin over a concentration range similar to that of the binding of biotinylated heparin to plate-bound clusterin. The binding of biotinylated heparin to clusterin was independent of the presence or absence of Ca2+. The binding of biotinylated heparin to plate-bound clusterin increased with decreasing pH over the range 5.5-8.0 and was characterized by an apparent pKa of 6.9. Clusterin in human serum bound to heparin-Sepharose at pH 6.0 but not at pH 7.4. Dot-blot experiments showed that one of the polypeptide chains of clusterin which had been reduced and alkylated under denaturing conditions bound to heparin-Sepharose. This chain was identified as the alpha chain from its N-terminal amino acid sequence.     

Identification and characterization of glycosylation sites in human serum clusterin

Clusterin is a ubiquitous, heterodimeric glycoprotein with multiple possible functions that are likely influenced by glycosylation. Identification of oligosaccharide attachment sites and structural characterization of oligosaccharides in human serum clusterin has been performed by mass spectrometry and Edman degradation. Matrix-assisted laser desorption ionization mass spectrometry revealed two molecular weight species of holoclusterin (58,505 +/- 250 and 63,507 +/- 200). Mass spectrometry also revealed molecular heterogeneity associated with both the alpha and beta subunits of clusterin, consistent with the presence of multiple glycoforms. The data indicate that clusterin contains 17-27% carbohydrate by weight, the alpha subunit contains 0-30% carbohydrate and the beta subunit contains 27-30% carbohydrate. Liquid chromatography electrospray mass spectrometry with stepped collision energy scanning was used to selectively identify and preparatively fractionate tryptic glycopeptides. Edman sequence analysis was then used to confirm the identities of the glycopeptides and to define the attachment sites within each peptide. A total of six N-linked glycosylation sites were identified, three in the alpha subunit (alpha 64N, alpha 81N, alpha 123N) and three in the beta subunit (beta 64N, beta 127N, and beta 147N). Seven different possible types of oligosaccharide structures were identified by mass including: a monosialobiantennary structure, bisialobiantennary structures without or with one fucose, trisialotriantennary structures without or with one fucose, and possibly a trisialotriantennary structure with two fucose and/or a tetrasialotriantennary structure. Site beta 64N exhibited the least glycosylation diversity, with two detected types of oligosaccharides, and site beta 147N exhibited the greatest diversity, with five or six detected types of oligosaccharides. Overall, the most abundant glycoforms detected were bisialobiantennary without fucose and the least abundant were monosialobiantennary, trisialotriantennary with two fucose and/or tetrasialotriantennary. Clusterin peptides accounting for 99% of the primary structure were identified from analysis of the isolated alpha and beta subunits, including all Ser- and Thr-containing peptides. No evidence was found for the presence of O-linked or sulfated oligosaccharides. The results provide a molecular basis for developing a better understanding of clusterin structure-function relationships and the role clusterin glycosylation plays in physiological function.     

Central nervous system cytokine mRNA expression following Theiler''s murine encephalomyelitis virus infection

This paper records the results of an investigation into potentiation and staircase phenomena in rightventricular guinea-pig papillary muscles with particular reference to the sarcoplasmic Ca(2+)-channel. As a tool to isolate the second ('late', 'tonic') component of isoproterenol-induced biphasic contractions ryanodine was used. On the evidence at present available the monophasic ryanodine-resistant component of the twitch represents that portion of the activator calcium which reaches the troponin C directly, that is, not taking the roundabout way through the intracellular storage structures. In order to avoid functional instabilities of the isolated muscle preparation a short-time double rest stimulation programme was used which combines a number of different tests and gives information on (1) the post-rest potentiation, (2) the post-extrasystolic potentiation, (3) the mechanical post-rest recovery, (4) the interval-strength relationship, and (5) the mechanical restitution. The results of the present work show that under the influence of ryanodine (1) the Bowditch staircase, a typical feature of normodynamic mammalian ventricular preparations as well as of hypodynamic frog heart preparations, does not exist, (2) the post-extrasystolic potentiation disappears, (3) the curve reflecting the mechanical restitution, under normal in vitro conditions a monotonically increasing function, becomes biphasic within the relative refractory period, (4) the conspicuous depression of the isometric post-rest contraction for long lasting pauses interrupting the regular pacing rhythm, a typical feature of isolated guinea-pig ventricular tissue, is clearly diminished, and (5) the characteristic curve, reflecting the potentiation of the post-extrasystolic post-rest contraction as a function of the delay time preceding the extrastimulus, becomes displaced to the premature interstimulus interval. The concept of an 'extended 2-calcium-store model' is supported by this work.     

Renal biopsy in lupus nephritis

Renal biopsy can be extremely valuable in the management of patients with lupus nephritis. It is remarkably common to find pathological evidence of substantial nephron loss in patients with low-grade laboratory abnormalities. This is due to compensatory hypertrophy and hemodynamic adjustments within the less-diseased nephron mass. It has been shown that the decision to institute immunosuppressive therapy is highly informed by the results of renal biopsy and offers the prospect of achieving more favorable renal outcomes. Kidney biopsies should be evaluated by dedicated renal pathology services experienced in diagnostic light, immunofluorescence and electron microscopy. Biopsies should be classified according to the World Health Organization (WHO) system and specific lesions semiquantitatively scored against a checklist of features comprising activity (reversible) and chronicity (irreversible damage) indices. The renal biopsy findings should be reviewed jointly by pathologists and the clinicians caring for patients with lupus nephritis.