Identification of a cis-acting element which assists in the recruitment of alternative transcription start sites/promoters.

The transcription start sites (TSSs) of the human plasma prekallikrein gene (KLKB1) determined in RNA from kidney are mostly localized within intron 1 and exon 2, suggesting that the region encompassing exon 1, intron 1, and exon 2 comprises an alternative promoter. Reporter gene analyses in HepG2 and immortalized human kidney epithelial cells confirmed a significant transcriptional activity of this putative promoter region. However, when the TSSs recruited for transcription of the reporter gene were determined, only a few transcripts starting within the insert were detected, whereas the majority of TSSs were located in the vector backbone up to about 2000 bp upstream of the insert. Further reporter gene studies with deletion mutants of the fragment exon 1-intron 1-exon 2 revealed that the 3'-terminal 13-bp segment of intron 1 is sufficient to promote transcriptional activity and induce upstream displacement of the TSS. We conclude that the 13-bp segment represents a cis-acting element which can displace TSSs by provoking the recruitment of alternative promoters and/or by masking intergenic transcription terminating signals.

Cellular expression of plasma prekallikrein in human tissues.

Plasma prekallikrein (PPK) is synthesised in hepatocytes and secreted into the blood, where it participates in the surface-dependent activation of blood coagulation, fibrinolysis, kinin generation and inflammation. Recently we demonstrated by quantitative RT-PCR that the human PPK gene is transcribed not only in the liver, but also in various non-hepatic human tissues at significant levels. However, up to now no reliable information is available concerning protein synthesis in the corresponding human tissues. Here we demonstrate by immunohistochemical studies that PPK or plasma kallikrein (PK) is localised in cells of different embryologically derived human tissues. In the human nephron, single cells of the distal tubules stained intensely, while the cytoplasm of cells forming proximal tubules and collecting ducts stained uniformly. PPK/PK was localised in hepatic epithelial cells of the liver, in cells of the pancreatic islet of Langerhans, in the interstitial Leydig cells of the testes, in the follicular and thecal granulosa cells of the ovary, and in the parotid gland, oesophagus, skin, respiratory tract, prostate and breast. We conclude that the cellular localisation of PPK/PK in multiple different progenitor-derived cells indicates specific cellular functions of this enzyme, in addition to its known function in the blood.

Kinin receptors in stimulated and characterized decidua tissue-derived cells.

Bradykinin and its kinin B(2) receptor are autocrine and paracrine mediators in foetal membranes and decidua. As a first step we characterized the intracellular morphology of decidual cells. Cultured decidua tissue-derived cells immunolabel for vimentin fibrils, and are considered to be of mesenchymal origin. They show characteristics of macrophages and can be distinguished from endothelial cells and cells of the trophoblast lineage. These cellular features were determined by means of immunocytochemistry. Furthermore cultured decidua tissue-derived cells express kinin B(2) receptors and in this context we demonstrated its expression at mRNA level by in situ reverse transcriptase polymerase chain reaction. Following stimulation with bacterial lipopolysaccharide, we have observed a marginal upregulation of the expression of kinin B(1) receptors and carboxypeptidase M by quantitative RT-PCR. Equilibrium binding experiments with [(3)H]des-Arg(10)-kallidin, the kinin B(1) receptor agonist, did not result in detectable binding sites.

Expression of the plasma prekallikrein gene: utilization of multiple transcription start sites and alternative promoter regions.

The plasma prekallikrein gene is expressed in many different human tissues at distinctly different levels and therefore tissue-specific control of the gene transcription is likely. In this study we demonstrate that transcription of the plasma prekallikrein gene can be initiated at multiple sites, for which at least four different promoters are utilized. A comparison of the genomic and mRNA sequences of mouse plasma prekallikrein revealed that the sequence segment that was formerly regarded as the first exon of the mouse plasma prekallikrein gene consists of three exons, with the first exon localized 14.2 kbp upstream of the translation start. For the rat and human plasma prekallikrein genes, in silico analysis suggested an analogous exon-intron organization. Determination of the transcription start sites showed that in both mouse and human, the proximal and distal regions could be utilized for transcription initiation; however, the proximal region is preferred. A deletion mutation analysis of the proximal promoter region using a 1.7-kbp segment revealed a strong activating region immediately upstream of the known mRNA, followed by both a modest repressor and an enhancer region.

Contact activation in low-density lipoprotein apheresis systems.

BACKGROUND: Anaphylactoid reactions due to contact activation have been observed in patients on ACE inhibitor therapy and hemodialysis with negatively charged dialysis membranes. Negatively charged surfaces are functional constituents of different LDL apheresis systems. Therefore, contact activation was investigated during LDL apheresis with three different systems: (i) heparin-induced extracorporeal LDL precipitation (HELP); (ii) dextran sulfate cellulose (DSC) columns; and (iii) modified polyacrylate gels (DALI) in a clinical setting. METHODS: 24 prevalent patients on regular LDL apheresis treatment were included in the study. Bradykinin, prekallikrein, and HMW kininogen were measured during a single LDL apheresis at different sites of the systems. RESULTS: LDL apheresis with DSC and DALI was associated with an extreme release of bradykinin after the passage of plasma or blood through the LDL adsorbers as well as with a decrease of prekallikrein and HMW kininogen during the course of the treatment. Bradykinin release exceeded the degradation capacity of the kininase II, since markedly elevated bradykinin concentrations were observed in the arterial line of the extracorporeal circuits of both systems. This was not associated with anaphylactoid reactions. In contrast to the treatments with DSC and DALI, the HELP system did not lead to any activation of the kallikrein-kinin system. CONCLUSION: From our data we conclude that angiotensin converting enzyme (ACE) inhibitors are contraindicated in patients on LDL apheresis with the DSC and the DALI system. Because the HELP system does not activate the kallikrein-kinin system, patients who need ACE inhibitors are predisposed for this LDL apheresis procedure.

Bradykinin and histamine generation with generalized enhancement of microvascular permeability in neonates, infants, and children undergoing cardiopulmonary bypass surgery.

OBJECTIVE: To investigate whether generation and liberation of bradykinin and histamine contribute to generalized edema formation in pediatric cardiopulmonary bypass surgery. DESIGN: Prospective observational study. SETTING: Pediatric heart surgery of a university hospital. PATIENTS: Forty-one neonates, infants, and children undergoing cardiopulmonary bypass to correct congenital cardiac anomalies. INTERVENTIONS: Plasma concentrations of bradykinin and histamine were determined before, during, and after cardiopulmonary bypass. Fluid balance was evaluated by control of fluid intake and output. MEASUREMENTS AND MAIN RESULTS: The susceptibility to generalized edema formation increased significantly (r = -.457; p <.005) with decreasing age. Approximately three times higher plasma concentrations of bradykinin (p <.001) were found at the onset of anesthesia and during the total observation period in patients with a fluid retention of >6% of body weight compared with patients with a lower retention rate. Plasma bradykinin reached significantly (p <.01) higher peak concentrations of 237.9 +/- 58.6 fmol/mL during cardiopulmonary bypass and of 227.5 +/- 90.7 fmol/mL during the early postoperative period in patients with severe edema formation in contrast to only 86.6 +/- 10.9 and 65.5 +/- 26.8 fmol/mL in patients with minor fluid retention. A tendency (p =.06) to slightly increasing histamine concentrations from 2.07 +/- 0.13 nmol/L at baseline to 3.32 +/- 1.41 nmol/L during 90 mins of cardiopulmonary bypass was only observed in patients with high fluid retention. CONCLUSIONS: Bradykinin seems to be essentially involved in the enhancement of microvascular permeability in pediatric cardiopulmonary bypass surgery, although a dominant causal role cannot be claimed by this study. Histamine, however, doesn't appear to play a major role and may only contribute as a cofactor. To what extent an increased expression of bradykinin-1 and bradykinin-2 receptors or a reduced potential of bradykinin-degrading enzymes is involved is the object of a further clinical study.

Mechanism of kinin release during experimental acute pancreatitis in rats: evidence for pro- as well as anti-inflammatory roles of oedema formation.

1 Kinin B(2) receptor antagonists or tissue kallikrein (t-KK) inhibitors prevent oedema formation and associated sequelae in caerulein-induced pancreatitis in the rat. We have now further investigated the mechanism of kinin generation in the pancreas. 2 Kinins were elevated in the pancreatic tissue already before oedema formation became manifest. Peak values (421+/-59 pmol g(-1) dry wt) were reached at 45 min and remained elevated for at least 2 h; a second increase was observed at 24 h. Pretreatment with the B(2) receptor antagonist icatibant abolished kinin formation, while post-treatment was ineffective. 3 Total kininogen levels were very low in the pancreas of controls, but increased 75-fold during acute pancreatitis. This increase was absent in rats that were pretreated with icatibant. 4 During pancreatitis, t-KK-like and plasma kallikrein (p-KK)-like activity in the pancreas, as well as trypsinogen activation peptide (TAP) increased significantly. Icatibant pretreatment further augmented t-KK about 100-fold, while p-KK was significantly attenuated; TAP levels remained unaffected. 5 Endogenous protease inhibitors (alpha(1)-antitrypsin, alpha(2)-macroglobulin) were low in normal tissues, but increased 45- and four-fold, respectively, during pancreatitis. This increase was abolished when oedema formation was prevented by icatibant. 6 In summary, oedema formation is initiated by t-KK; the ensuing plasma protein extravasation supplies further kininogen and active p-KK to the tissue. Concomitantly, endogenous protease inhibitors in the oedema fluid inhibit up to 99% of active t-KK. Our data thus suggest a complex interaction between kinin action and kinin generation involving positive and negative feedback actions of the inflammatory oedema.

The hyaluronan-binding serine protease from human plasma cleaves HMW and LMW kininogen and releases bradykinin.

The influence of the hyaluronan-binding protease (PHBSP), a plasma enzyme with FVII- and pro-urokinase-activating potency, on components of the contact phase (kallikrein/kinin) system was investigated. No activation or cleavage of the proenzymes involved in the contact phase system was observed. The pro-cofactor high molecular weight kininogen (HK), however, was cleaved in vitro by PHBSP in the absence of any charged surface, releasing the activated cofactor and the vasoactive nonapeptide bradykinin. Glycosoaminoglycans strongly enhanced the reaction. The cleavage was comparable to that of plasma kallikrein, but clearly different from that of coagulation factor FXIa. Upon extended incubation with PHBSP, the light chain was further processed, partially removing about 60 amino acid residues from the N-terminus of domain D5 of the light chain. These cleavage site(s) were distinct from plasma kallikrein or FXIa cleavage sites. PHBSP and, more interestingly, also plasma kallikrein could cleave low molecular weight kininogen in vitro, indicating that domains D5H and D6H are no prerequisite for kininogen cleavage. PHBSP was also able to release bradykinin from HK in plasma where the pro-cofactor circulates predominantly in complex with plasma kallikrein or FXI. In conclusion, PHBSP represents a novel kininogen-cleaving and bradykinin-releasing enzyme in plasma that shares significant catalytic similarities with plasma kallikrein. Since they are structurally unrelated in their heavy chains (propeptide), their similar in vivo catalytic activities might be directed at distinct sites where PHBSP could induce processes that are related to the kallikrein/kinin system.

Eluate derived by extracorporal antibody-based immunoadsorption elevates the cytosolic Ca2+ concentration in podocytes via B2 kinin receptors.

BACKGROUND/AIM: Patients with idiopathic focal segmental glomerulosclerosis (FSGS) often develop a recurrence of the disease after kidney transplantation. In a number of FSGS patients, plasmapheresis and immunoadsorption procedures have been shown to transiently reduce proteinuria and are thought to do this by eliminating a circulating factor. Direct cellular effects of eluates from immunoadsorption procedures on podocytes, the primary target of injury in FSGS, have not yet been reported. METHODS: Eluates were derived from antibody-based immunoadsorption of a patient suffering from primary FSGS, a patient with systemic lupus erythematosus, and a healthy volunteer. The cytosolic free Ca(2+) concentration ([Ca(2+)](i)) of differentiated podocytes was measured by single-cell fura-2 microfluorescence measurements. Free and total immunoreactive kinin levels were measured by radioimmunoassay. RESULTS: FSGS eluates increased the [Ca(2+)](i) levels concentration dependently (EC(50) 0.14 mg/ml; n = 3-19). 1 mg/ml eluate increased the [Ca(2+)](i) values reversibly from 82 +/- 12 to 1,462 +/- 370 nmol/l, and then they returned back to 100 +/- 16 nmol/l (n = 19). The eluate-induced increase of [Ca(2+)](i) consisted of an initial Ca(2+) peak followed by a Ca(2+) plateau which depended on the extracellular Ca(2+) concentration. The eluate-induced increase of [Ca(2+)](i) was inhibited by the specific B(2) kinin receptor antagonist Hoe 140 in a concentration-dependent manner (IC(50) 2.47 nmol/l). In addition, prior repetitive application of bradykinin desensitized the effect of eluate on [Ca(2+)](i). A colonic epithelial cell line not reacting to bradykinin did not respond to eluate either (n = 6). Similar to FSGS eluates, the eluate preparations of both the systemic lupus patient and the healthy volunteer led to a biphasic, concentration-dependent [Ca(2+)](i) increase in podocytes which again was inhibited by Hoe 140. Free kinins were detected in all eluate preparations. CONCLUSION: The procedure of antibody-based immunoadsorption leads to kinin in the eluate which elevates the [Ca(2+)](i) level of podocytes via B(2) kinin receptors.