Bradykinin-induced inhibition of cell proliferation and tyrosine kinase activity in rat mesangial cells.

The relationship between cell proliferation, protein tyrosine phosphorylation, phosphotyrosine kinase activity and bradykinin receptor activation in rat mesangial cells was investigated. We demonstrated that bradykinin (BK), through the B2 receptor, induced a dose-dependent inhibition of mesangial cell proliferation stimulated by fetal calf serum. We next found that BK induced a dose-dependent inhibition of phospho-tyrosine kinase activity. Treatments with pertussis-toxin, inhibition of phospholipase C and protein kinase C inhibitors and chelation of free cytosolic calcium did not change the bradykinin-induced inhibition of phosphotyrosine kinase. Western blot analysis of phosphotyrosinated proteins demonstrated that BK reduced tyrosine phosphorylation of several proteins among which we identified the 125-focal adhesion kinase. Taken together, these data suggest for the first time that, in proliferating rat mesangial cells, B2 receptor stimulation is able to induce, via a pertussis insensitive pathway, the inhibition of tyrosine kinase activity and mesangial cell proliferation.

Homologous and heterologous induction of the human bradykinin B1-receptor and B1-receptor localisation along the rat nephron.

The kinin B1-receptor which is absent or expressed at very low levels under physiological conditions is strongly induced under inflammatory conditions. It has been shown that B1-receptor induction during inflammation involves interleukin-1beta (IL-1beta) production and activation of nuclear factor-kappaB (NF-kappaB). Since bradykinin (BK), the B2-receptor agonist induces IL-1beta expression and activates NF-kappaB, we have analysed the effect of B2-receptor activation in cultured human lung fibroblasts cells on B1-receptor expression by a semiquantitative RT-PCR analysis. Treatment with BK resulted in a significant increase in the expression of B1-receptor mRNA which was abolished by a specific B2-receptor antagonist. This result suggests that B2-receptor activation can prime the expression of B1-receptors. Although the renal localisation of the B2-receptor has been thoroughly studied, nothing is known about the distribution of the B1-receptor in the kidney. Using a combination of microdissection and a semiquantitative RT-PCR/Southern blot analysis we showed the absence of B1-receptors under physiological conditions in 10 microdissected rat nephron segments. However, 18 h LPS-treatment induced significant expression of the B1-receptor in all, but one segment. These studies provide the first molecular basis for the observed changes in renal haemodynamics after B1-agonist infusion in animal kidney models.

Effect of bradykinin on tyrosine kinase and phosphatase activities and cell proliferation in mesangial cells.

We investigated the relationship between protein tyrosine phosphorylation and bradykinin (BK) receptor activation in rat mesangial cells (MC). Stimulation of the B2 receptor resulted in a dual effect consisting of an independent activation and inhibition of tyrosine kinase activity (TKA). The activation was rapid and transient, reaching a peak value at 30 s whereas the inhibition was observed at 5 min and persisted up to 10 min. Treatments with pertussis-toxin and U73122 showed that only the BK-induced stimulation of TKA is dependent on phospholipase C activation via a pertussis-toxin sensitive G-protein. In addition, BK induced an increase in tyrosine phosphatase activity. Western-blot analysis demonstrated that the dual effect of BK on TKA was associated with both an increase and a decrease in tyrosine phosphorylation of the p125-focal adhesion kinase (FAK). Moreover, BK was able to reduce the maximal stimulated MC cell proliferation induced by fetal calf serum. These data show that in rat MC, B2 receptor stimulation activates and inhibits two independent tyrosine kinase signaling pathways associated with tyrosine phosphorylation of p125-FAK that might be implicated in MC proliferation.

Inhibition of cGMP accumulation in mesangial cells by bradykinin and tyrosine kinase inhibitors.

We examined the effect of bradykinin (BK) on the accumulation of cGMP of the mesangial cell (MC), a smooth muscle-like cell of the renal glomerulus. BK caused a time- and concentration dependent reduction of the cGMP concentration. In addition, BK inhibited total protein tyrosine kinase (PTK) activity. Two tyrosine kinase inhibitors (TKI) genistein and tyrphostin also reduced the cGMP concentration. The inhibition of BK and TKI were not additive. The inhibition of PTK by BK, mediated through activation of the B2-receptor, was unaffected by inhibitors of Gi/o proteins, phospholipase C, protein kinase C, cyclooxygenase and Ca2+ release from intracellular stores. Only IBMX a broad spectrum inhibitor of phosphodiesterases (PDE) and 8-methoxymethyl IBMX a specific type-1 PDE inhibitor prevented the inhibitory effects of BK and TKI indicating the involvement of type-1 PDE. In addition, BK had no effect on soluble guanylate cyclase (sGC) and nitric oxide synthase activity. In freshly isolated glomeruli, which represent the physiological environment of MC, BK also reduced the cGMP concentration. Like in MC, the inhibitory effect was suppressed by IBMX. These data demonstrate that BK suppresses a PTK-dependent pathway of cGMP production in rat MC at a level downstream of NO synthase and sGC. It is suggested that BK and TKI inhibitors decrease cGMP levels by preventing tyrosine phosphorylation of type-1 PDE activity, thereby leading to enzyme activation.

Renal bradykinin receptors: localisation, transduction pathways and molecular basis for a possible pathological role (review).

Kinins are biologically active peptides that exert their effects by activating two seven transmembrane G-protein coupled receptors termed B1 and B2 which have only about 36% of homology. The major kinin peptide under physiological conditions, bradykinin (BK), modulates renal haemodynamics and function. Under physiological conditions most BK effects involve bradykinin B2-receptors. Studies on the intra-cellular transduction pathways, the regulation of the expression and the localisation of these receptors along the nephron, as well as the first studies on transgenic mice models, have allowed to better define the role of these receptors under physiological and pathological conditions. The role of the renal B1-receptor, induced in a variety of pathologies related to inflammation, is poorly understood. Recent investigations on the molecular mechanism of B1-receptor induction and its detailed renal localisation have shown that under inflammatory conditions this kinin receptor might be of importance. B2-receptors are suggested to be involved in part of the renoprotective effects of angiotensin converting enzyme (ACE)-inhibitors in insulin-dependent diabetes. However, ACE-inhibitor treatment, resulting also in an increased B1-agonist concentration might result in homologous induction and activation of the B1-receptor.

Determination of methotrexate and 7-hydroxymethotrexate by liquid chromatography for routine monitoring of plasma levels.

A high-performance liquid chromatographic (HPLC) method was designed to meet analytical and metrological requirements for routine blood level monitoring of methotrexate (MTX) and its main metabolite 7-hydroxymethotrexate (7OMTX). The metabolite, unavailable as a pure substance, was measured by reference to MTX calibration according to their respective ultraviolet absorbances. Acetonitrile deproteinization and chloroform clean-up provided plasma samples devoid of long-retained contaminants. The precision of the HPLC measurements, reproducibility of clean-up recovery, matrix effects and linearity were assessed by analysis of variance and linear regression in an appropriate experimental design, within a range from 0.205 to 16.7 mg/l of MTX and from 0.084 to 6.83 mg/l of 7OMTX. The clean-up recovery from plasma was 88% for MTX and 72% for 7OMTX, owing to retention on the protein precipitate. The assay was linear, the measurement precision was 3.3% for MTX and 6.2% for 7OMTX and the clean-up reproducibility was 4% for MTX and 3.6% for 7OMTX. By reference to automated fluorescence polarization immunoassay, the HPLC method resulted in plasma MTX values 10% lower, probably owing to the higher specificity of HPLC. Unsystematically sequenced plasma samples from 35 children following 24-h MTX infusions provided estimated half-decay times of 16 and 19 h for MTX and 7OMTX, respectively, and 7OMTX:MTX concentration ratios of 7 at 48 h and of 5 at 72 h from starting infusions.