Delayed blockade of the kinin B1 receptor reduces renal inflammation and fibrosis in obstructive nephropathy.

Renal fibrosis is the common histological feature of advanced glomerular and tubulointerstitial disease leading to end-stage renal disease (ESRD). However, specific antifibrotic therapies to slow down the evolution to ESRD are still absent. Because persistent inflammation is a key event in the development of fibrosis, we hypothesized that the proinflammatory kinin B1 receptor (B1R) could be such a new target. Here we show that, in the unilateral ureteral obstruction model of renal fibrosis, the B1R is overexpressed and that delayed treatment with an orally active nonpeptide B1R antagonist blocks macrophage infiltration, leading to a reversal of the level of renal fibrosis. In vivo bone marrow transplantation studies as well as in vitro studies on renal cells show that part of this antifibrotic mechanism of B1R blockade involves a direct effect on resident renal cells by inhibiting chemokine CCL2 and CCL7 expression. These findings suggest that blocking the B1R is a promising antifibrotic therapy.

The B1-agonist [des-Arg10]-kallidin activates transcription factor NF-kappaB and induces homologous upregulation of the bradykinin B1-receptor in cultured human lung fibroblasts.

The bradykinin B1-receptor is strongly upregulated under chronic inflammatory conditions. However, the mechanism and reason are not known. Because a better understanding of the mechanism of the upregulation will help in understanding its potential importance in inflammation, we have studied the molecular mechanism of B1-receptor upregulation in cultured human lung fibroblasts (IMR 90) in response to IL-1beta and the B1-agonist [des-Arg10]-kallidin. We show that treatment of human IMR 90 cells by IL-1beta stimulates the expression of both B1-receptor mRNA and protein. The latter was studied by Western blot analysis using antipeptide antibodies directed against the COOH-terminal part of the human B1-receptor. We furthermore report the novel observation that the B1-receptor is upregulated by its own agonist which was completely blocked by the specific B1-antagonist [des-Arg10-Leu9]-kallidin, indicating an upregulation entirely mediated through cell surface B1-receptors. The increased population of B1-receptors was functionally coupled as exemplified by an enhancement of the B1-agonist induced increase in free cytosolic calcium. Upregulation by the B1-agonist was blocked by a specific protein kinase C inhibitor. B1-agonist-induced upregulation was correlated to the induction of transcription factor nuclear factor kappaB (NF-kappaB) which efficiently bound to the NF-kappaB-like sequence located in the promoter region of the human B1-receptor gene. This correlation was further confirmed by reporter gene assays which showed that this NF-kappaB-like sequence, in the B1-receptor promoter context, could contribute to IL-1beta and DLBK-induced B1-receptor transcription activation, and by the effect of NF-kappaB inhibitor pyrrolidinedithiocarbamate which diminished both B1-receptor upregulation and NF-kappaB activation. NF-kappaB is now recognized as a key inflammatory mediator which is activated by the B1-agonist but which is also involved in B1-receptor upregulation.

Role of tissue kallikrein in the cardioprotective effects of ischemic and pharmacological preconditioning in myocardial ischemia.

Tissue kallikrein (TK), a major kinin-forming enzyme, is synthesized in the heart and arteries. We tested the hypothesis that TK plays a protective role in myocardial ischemia by performing ischemia-reperfusion (IR) injury, with and without ischemic preconditioning (IPC) or ACE inhibitor (ramiprilat) pretreatment, in vivo in littermate wild-type (WT) or TK-deficient (TK-/-) mice. IR induced similar infarcts in WT and TK-/-. IPC reduced infarct size by 65% in WT, and by 40% in TK-/- (P<0.05, TK-/- vs WT). Ramiprilat also reduced infarct size by 29% in WT, but in TK-/- its effect was completely suppressed. Pretreatment of WT with a B2, but not a B1, kinin receptor antagonist reproduced the effects of TK deficiency. However, B2 receptor-deficient mice (B2-/-) unexpectedly responded to IPC or ramiprilat like WT mice. But pretreatment of the B2-/- mice with a B1 antagonist suppressed the cardioprotective effects of IPC and ramiprilat. In B2-/-, B1 receptor gene expression was constitutively high. In WT and TK-/- mice, both B2 and B1 mRNA levels increased several fold during IR, and even more during IPC+IR. Thus TK and the B2 receptor play a critical role in the cardioprotection afforded by two experimental maneuvers of potential clinical relevance, IPC and ACE inhibition, during ischemia.

Compared effects of a low and a high sodium diet on the renal and urinary concentration and activity of kallikrein in normal rats.

Different sodium intakes may affect or alter the urinary excretion of renal kallikrein. We have compared the renal and the urinary effects of sodium depletion and sodium loading with 1% NaCl on total kallikrein, using a direct radio-immunoassay against immunoreactive kallikrein and on active kallikrein assessed by a kininogenase assay with a kinin radio-immunoassay. Sodium depletion resulted in an increase in renal and urinary excretion of both the immunoreactive kallikrein and the kininogenase activity. Sodium loading resulted in a slight but significant decrease in renal tissue immunoreactive kallikrein content without any change in the kininogenase activity, while the urinary excretion of the kininogenase activity was greatly increased and the urinary immunoreactive kallikrein remained steady. This sodium depletion induces consistent effects at the renal and urinary level, resulting probably from a stimulation of biosynthesis. However, during sodium loading, renal and urinary measurements of kallikrein are dissociated. Furthermore, kininogenase activity and immunoreactive kallikrein are not correlated in either of the two studied compartments. Thus sodium loading seems to induce independent effects at the renal and urinary levels, possibly resulting from different mechanisms.

Localization of a renal kallikrein immunoreactive-like substance in rat ureter.

An antibody against rat kallikrein was produced in rabbits and its localization was studied in various organs of the rat to confirm its specificity. The distribution of immunoreactive kallikrein was studied in rat ureter by use of immunochemical techniques. Ureteral tissue was fixed in Zamboni's-glutaraldehyde fixative and immunostained with indirect immunofluorescence and the peroxidase-antiperoxidase (PAP) method for light and electron microscopy. Preabsorption of the primary polyclonal antiserum with purified rat urinary kallikrein and substitution with normal serum were used as controls. By light microscopy, kallikrein was localized in the lamina propria and in the adventitial connective tissue surrounding the entire ureter. Immunoelectron microscopy confirmed this immunolocalization. Immunoreactive kallikrein was concentrated in fibroblasts of connective tissue and was not present in collagen fibers. Immunoreactivity was associated with the Golgi complex, free polyribosomes, and rough endoplasmic reticulum. No immunostaining was observed in other subcellular components of fibroblasts.

Yohimbine increases submaxillary kallikrein release into the saliva in dogs: evidence for alpha 2-adrenoceptor-mediated inhibition of cholinergic pathways.

1. The effects of the alpha 2-adrenoceptor antagonist, yohimbine (0.5 mg kg-1, i.v.) on basal, sympathetic and parasympathetic stimulation-induced submaxillary kallikrein release were investigated in the anaesthetized dog. Kallikrein was measured by its kininogenase activity before and after trypsin activation which also allowed a study of the proportion of active to total enzyme. 2. Yohimbine induced a rapid, three fold increase in basal kallikrein release correlated with an increase in salivary flow rate which lasted for 60 min following injection. 3. Sectioning the chorda tympani did not affect basal kallikrein release but abolished yohimbine-induced rise in salivary kallikrein secretion. 4. Parasympathetic stimulation alone induced a 3 to 4 fold increase in basal kallikrein release correlated with an increase in salivary flow rate. Yohimbine induced a significant additional increase in parasympathetic-stimulated kallikrein release. 5. When the cervical sympathetic nerve was sectioned the basal kallikrein release decreased by 30 to 40%. 6. Sympathetic stimulation alone also induced a 3 to 4 fold increase in basal kallikrein. This was not correlated with the salivary flow and unaffected by yohimbine. 7. The results indicate that yohimbine increases submaxillary kallikrein release into the saliva by inhibition of presynaptic alpha 2-adrenoceptors located on the chorda tympani nerve endings.

Cyclosporine A-induced increase in glomerular cyclic GMP in rats and the involvement of the endothelinB receptor.

1. A transient two fold increase in the cyclic GMP content was observed in rat freshly isolated glomeruli 6 to 9 h after a single subcutaneous injection of 20 mg kg-1 cyclosporine A (CsA) in conscious animals. 2.In vitro stimulation with endothelin 3 (ET-3) of isolated glomeruli obtained from CsA-untreated rats resulted in a dose-dependent increase in cyclic GMP content. The increase observed with 10 nM ET-3 was similar to that observed in glomeruli isolated 9 h after in vivo CsA administration. 3. The rise in glomerular cyclic GMP content after in vivo CsA injection was prevented by in vivo treatment with L-NAME (10 mg kg-1) or by in vitro calcium deprivation of the incubation medium. 4. The stimulating effects of CsA on glomerular cyclic GMP content were inhibited by in vivo administration of the ETB receptor antagonist BQ-788 (2 mg kg-1) but not by the ETA receptor antagonist BQ-123 (2 mg kg-1). 5. The maximum increase in glomerular cyclic GMP content induced in vitro by acetylcholine (100 microM) and by ET-3 (100 nM) was slightly lower (approximately by 20-25%; P < 0.05) in glomeruli from CsA-treated rats than in glomeruli from untreated rats. In contrast, the maximum increase achieved with 1 microM sodium nitroprusside was similar in both groups. 6. A single subcutaneous injection of CsA did not significantly alter the glomerular mRNA expression of constitutive endothelial NO synthase (eNOS), as evaluated by RT-PCR, whereas the mRNA expression of the inducible NO synthase (iNOS), which follows pretreatment with lipopolysaccharide, was prevented. 7. These results indicate that in vivo administration of a single dose of cyclosporine A transiently increases the cyclic GMP content of freshly isolated glomeruli, and that activation of ETB receptors and stimulation of the NO pathway are involved in this process. Furthermore, a single administration of CsA does not impair eNOS mRNA expression and only slightly reduces NO-dependent glomerular cyclic GMP production.

Bradykinin stimulates production of inositol (1,4,5) trisphosphate in cultured mesangial cells of the rat via a BK2-kinin receptor.

1. Using [125I-Tyr0]-BK, as radiolabelled ligand, and various agonists and antagonists of bradykinin (BK) we identified a single class of specific BK2-binding sites in mesangial cell membranes (Bmax = 73 fmol mg-1 protein and Kd = 3.7 nM). 2. Following the addition of 0.1 microM BK, inositol (1,4,5) trisphosphate (IP3) formation increased within 20 s from a basal level of 64 to a maximal value of 175 pmol mg-1 protein. 3. Incubation in a Ca(2+)-free medium did not change IP3 production but a 5 min preincubation with 1 mM EGTA completely prevented the BK-induced IP3 formation, suggesting that IP3 formation is partly dependent on extracellular calcium. 4. The BK2 antagonist D-Arg-Hyp3-D-Phe7-BK (10 microM) but not the BK1 antagonist (des-Arg9-Leu8-BK) abolished IP3 production in response to 0.1 microM BK. Pretreatment of mesangial cells with pertussis toxin was without effect on BK-induced IP3 formation, whereas phorbol 12-myristate 13-acetate significantly enhanced (by 25%) BK-induced IP3 formation. 5. The present data demonstrate that inositol phosphate breakdown in rat mesangial cells can be mediated via activation of a BK2-kinin receptor and is under negative control of protein-kinase C.

Intracellular Ca2+ depletion and Ca2+ channel blockers increase renal kallikrein secretion.

This study examined the effect of various manipulations of intracellular Ca2+ on kallikrein release by renal cortical slices. Increasing the extracellular Ca2+ concentration and the addition of Ca2+ ionophore A23187 was without effect on kallikrein release. In contrast, kallikrein release was enhanced by the addition of either extracellular or intracellular Ca2+ chelators in Ca(2+)-free medium and by two Ca2+ channel blockers, verapamil and nifedipine. Kallikrein release was also highly enhanced in depolarising medium (10-100 mM potassium chloride). Since potassium chloride induced a dose-related increase in free cytosolic Ca2+ which was abolished by nifedipine whereas the stimulation of kallikrein secretion persisted, a direct stimulating effect of potassium, at least at sub-physiological concentration, is suggested. Similarily, inhibition of either sodium/potassium-ATPase and Ca2+ ATPase by ouabain and vanadium respectively, was also without effect on kallikrein secretion. Taken together, these results indicate that intracellular Ca2+ depletion, Ca2+ channel blockers and high extracellular K+ concentrations, acting through different mechanisms, are effective stimuli for kallikrein secretion, at least in the isolated renal cortical slice.

In vivo and in vitro homologous desensitization of rat glomerular bradykinin B2 receptors.

We investigated the effects of bradykinin on glomerular bradykinin B2 receptor functions and parameters in vivo, after intrarenal infusion of bradykinin, and in vitro, after incubation of isolated rat glomeruli with bradykinin. Bradykinin transiently increased renal plasma flow whereas a second challenge was ineffective. Scatchard analysis demonstrated the presence of two populations of bradykinin binding sites whose densities were similarly decreased by about 40% after intrarenal bradykinin infusion. This decrease was not altered by an acid wash suggesting internalization of the radiolabelled ligand. The effect of bradykinin was prevented by a bradykinin B2 receptor antagonist. Pre-exposure of isolated rat glomeruli to bradykinin mimicked the in vivo results because there was a reduction in bradykinin-induced prostaglandin E2 and prostaglandin F2 alpha release. Rapid recovery was observed 15 min after washing out the bradykinin. Our results directly demonstrate a negative homologous down-regulation of B2 glomerular bradykinin receptor density under both in vivo and in vitro conditions, an effect which involves a rapid sequestration of the receptor.

Characterization of a B2-bradykinin receptor in rat renal mesangial cells.

The specific binding of bradykinin (BK) was investigated using membrane fractions from mesangial cells in primary culture, a cloned cell line, and in intact adherent cells with three different radiolabelled BK analogues: 125I-[Tyr0]BK, 125I-[Tyr5]BK and 125I-[Tyr8]BK. The best radioligand was 125I-[Tyr0]BK, and assay conditions were determined to ensure maximal stable binding. Binding appeared to be reversible and not to be inhibited by a wide variety of protease inhibitors including converting enzyme inhibitor and phosphoramidon. The maximum density of binding sites (Bmax) was about 88 +/- 18 fmol/mg protein, which is equivalent to about 6000 sites/cell, and the dissociation constant averaged 2 nM. No significant difference in Bmax was observed between membranes from cells in primary culture and those from cloned cells. Of the BK analogues tested, unmodified BK exhibited the highest inhibition constant (close to 10(-10) M). No displacement of 125I-[Tyr0]BK was observed in the presence of the B1 agonist des-Arg9-BK or several unrelated peptides, including atrial natriuretic factor and angiotensin I and II, whereas 50% inhibition of binding was achieved with the B2 antagonist [D-Arg,Hyp3,D-Phe7]BK (10(-9)M). Addition of BK for 3 min to the incubation medium of cloned mesangial cells induced a dose- and time-dependent increase in PGE2 unlike des-Arg9-BK, which showed no such effect. The secretion was strongly inhibited by prior incubation with the B2 antagonist [D-Arg,Hyp3,D-Phe7]BK. The pharmacological profile of the binding site determined with various BK agonists and antagonists, and the stimulating effect of binding site activation on prostaglandin release strongly suggest that B2-kinin-like receptors are present in rat mesangial cells.

Rapid automated analysis of glutathione reductase, peroxidase, and S-transferase activity: application to cisplatin-induced toxicity.

We describe a rapid kinetic method for the automated determination of the xenobiotic-metabolizing enzymes glutathione reductase, glutathione peroxidase and glutathione S-transferase, and its application to the study of cisplatin-induced toxicity. Liver, kidney and urine from control and cisplatin-treated rats were used as the source of enzymes. Advantages over conventional spectrophotometric methods include speed (25 assays in 4 min), small sample size, and improved precision. We show that glutathione S-transferase activity in liver is slightly reduced by cisplatin treatment, whereas all three enzymes are reduced in the kidney. Glutathione-S-transferase activity appeared in urine between the third and seventh days after cisplatin injection. Using these enzyme activities in cisplatin-treated rats, we suggest that the renal enzymes are more sensitive markers of toxicity than hepatic enzymes.

Effects of lysophosphatidic acid on proliferation and cytosolic Ca++ of human adult vascular smooth muscle cells in culture.

Lysophosphatidic acid (LPA) is a lipid mediator generated by activated platelets and having various effects on numerous cell types. We investigated some effects of 1-oleyl LPA on vascular smooth muscle cells cultured from adult human normal arteries. At micromolar concentrations, LPA induced a mitogenic effect ([3H]-thymidine incorporation and cell proliferation) on quiescent cells, without an additional growth factor being required. This effect was equipotent to that of 10% fetal calf serum, and it was accompanied by early (5 minutes) and late (1-3 hours) phosphorylation of mitogenactivated protein kinase. LPA inhibited cell migration through collagen coated membranes, with or without platelet-derived growth factor BB as chemoattractant. LPA induced a typical biphasic Ca2+ signal response made up of a rapid first phase due to Ca2+ release from intracellular stores followed by a second wave due to external Ca2+ influx. These findings support the proposal that LPA released from activated platelets is a mediator for smooth muscle cell response at the site of vessel injury in humans.

Renal kallikrein excretion as a distal nephrotoxicity marker during cadmium exposure in rats.

Cadmium exposure is known to induce hypertension, but development of hypertension is not universal in exposed animals. However, the cellular uptake of cadmium could also exert renal cytotoxic effects which have been, until now, essentially only studied at the proximal tubule level. Kallikrein is an enzyme synthetized in renal cortex and excreted in the urine in the distal tubule. Therefore, to evaluate the distal renal effect of cadmium, we studied the daily urinary kallikrein excretion (UKE) in conscious unrestrained female Brown Norway rats during long-term chronic exposure to 2 dosages of cadmium given subcutaneously 3 times a week, a low dose (LD): 0.25 mg/kg and a high dose (HD): 1 mg/kg. Neither dose of cadmium was able to induce significant hypertension in the treated animals. HD administration for 24 weeks resulted in a decreased UKE associated with an increase in plasma renin activity and sodium and potassium excretions. LD administration had no significant effect on UKE. Twenty weeks after stopping cadmium administration, a persistent reduction in UKE was still observed; furthermore, the group which had been previously administered a LD of cadmium, now also exhibited a reduced UKE. During this re-examination period in both groups, the UKE reductions were associated with normal systolic blood pressure, glycosuria, natriuresis. Our data show that cadmium administration can influence UKE, plasma renin activity, plasma aldosterone concentration and electrolyte excretion without inducing any variation of blood pressure. This may reflect a nephrotoxic, non-hypertensive effect. Since this effect persisted after stopping cadmium administration, it may indicate a prolonged irreversible nephrotoxic effect at the distal nephron level. Thus, UKE may be a useful non-invasive index to evaluate distal nephrotoxicity.

Effects of cicletanine on vasoactive systems in conscious sinoaortic-denervated dogs.

The present study investigates the antihypertensive action of cicletanine, a new antihypertensive compound with diuretic properties (or placebo), on vasopressor (catecholamines, renin-aldosterone) as well as vasodepressor (prostaglandins, kallikrein-kinin) systems in conscious chronic sinoaortic denervated (SAD) dogs. Cicletanine (10 mg/kg twice a day, per os, for one month) lowered blood pressure and heart rate. The antihypertensive action does not involve an effect on sympathetic tone (since plasma catecholamine levels were unmodified) or on plasma aldosterone levels. By contrast, urinary 6 keto PGF1 or PGE2 levels and kallikrein activity were enhanced. This result indicates that the antihypertensive effect of cicletanine is associated with a stimulation of potential vasodepressor systems (such as prostaglandins or kinins).

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.

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.

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.

Change in cortical active kallikrein during the onset of Goldblatt hypertension in the rat.

In two-kidney, one clip hypertensive rats renal cortical kallikrein was studied one and two weeks following induction of the hypertension in comparison with sham time controls. Cortical active kallikrein was lower in hypertensive rats only at week 2, although blood pressure was significantly higher one week after clipping. Total cortical kallikrein was however never different in sham control and in hypertensive rats. This results provide evidence for a change in the activation of prekallikrein during the onset of Goldblatt hypertension.

Hydrolysis of rat high molecular weight kininogen by purified rat urinary kallikrein: identification of bradykinin as the kinin formed.

We have previously reported that, although human urinary kallikrein, like glandular kallikreins for other species, releases lysyl-bradykinin from homologous and heterologous substrates, rat urinary kallikrein released a kinin which migrated like bradykinin in CM-cellulose chromatography and polyacrylamide gel electrophoresis (BBA677, 471, 1981). In the study we definitively established the nature of the kinin produced by rat urinary kallikrein by using purified enzyme and substrate, HPLC, radioimmunoassay and N-terminal analysis. Rat urinary kallikrein was purified to apparent homogeneity by a procedure which included affinity chromatography on aprotinin agarose. The kinin produced by rat urinary kallikrein acting on either pure rat high molecular weight kininogen or rat plasma or semipurified bovine and dog plasma was identified as bradykinin. This observation provides the evidence of species differences in the specificity of glandular kallikreins acting on kininogens.