Monoamine oxidase in developing rat renal cortex: effect of dexamethasone treatment.

The expression of the biogenic amine degrading enzyme monoamine oxidases-A and -B depends on several factors including regional distribution, development and hormonal environment. In the present study, we investigated the expression of monoamine oxidases in developing kidney and their regulation by dexamethasone treatment. Immunoblots and enzyme assays, performed using [14C]5-hydroxytriptamine and [14C]beta-phenylethylamine as substrates for monoamine oxidases-A and -B, respectively, showed that monoamine oxidase-A is the isoenzyme largely predominant in 9-day-old rats renal cortex. Experiments performed in 5-week-old rats showed an increase in monoamine oxidase-B activity and a decrease in monoamine oxidase-A activity and substrate affinity. The changes of monoamine oxidase-A activity and affinity were mimicked by dexamethasone treatment (0.60 mg/kg body weight injected subcutaneously three times at intervals of 24 h) of 9-day-old rats. In contrast, dexamethasone administration induced a modification of monoamine oxidase-B activity opposite to that found between 9-day- and 5-week-old rats. Dexamethasone treatment did not modify immunoreactivity and mRNA corresponding to monoamine oxidases-A and -B indicating that changes of enzyme activities were unrelated to regulation of protein synthesis and mRNA turnover. These results show that monoamine oxidases-A and -B are differently expressed in developing renal cortex and are regulated by dexamethasone treatment.

Endothelin increases NO-dependent cGMP production in isolated glomeruli but not in mesangial cells.

The ability of endothelins (ETs) to modulate nitric oxide-dependent glomerular guanosine 3'-5'-cyclic monophosphate (cGMP) production has recently been reported. The aim of this study was to directly confirm, using an antagonist, the involvement of the ETB receptor subtype and to investigate the potential role of mesangial cells (MC) in this ET-induced cGMP production. In glomeruli freshly isolated from rats, endothelin-3 (ET-3) induced a dose-dependent increase in cGMP content. This increase was inhibited by NG-monomethyl-L-arginine (L-NMMA) and methylene blue and was calcium dependent. Moreover, the effect of ET-3 was prevented by two ETB-selective receptor antagonists, BQ-788 and IRL-1038, but not by BQ-123, an ETA-selective receptor antagonist. It therefore appeared that ET-3 stimulates the glomerular constitutive NO pathway through activation of the ETB receptor subtype. In contrast, ET-3 and calcium ionophore had no effect on cGMP formation in cultured MC, whereas incubation with sodium nitroprusside resulted in an approximately 50-fold increase in the intracellular content of cGMP. However, ET-3 induced a dose-dependent rise in free MC cytosolic calcium that was abolished by an ETB antagonist. Moreover, both ETA and ETB receptors mRNA were expressed in primary cultures of MC. Finally, we failed to detect the presence of constitutive NO synthase (NOS), as demonstrated by the absence of L-citrulline forming activity and of the mRNA encoding for endothelial NOS, whereas they were present in isolated glomeruli. These data indicate that MC, despite the fact that they express ETB receptors, are not involved in glomerular NO production induced by exposure to ET-3, because they do not express constitutive NO synthase.

RT-PCR microlocalization of bradykinin B2 receptor mRNA in microdissected rat nephron segments.

Microlocalization of mRNA coding for the bradykinin 2 (BK2) receptor was carried out in the rat kidney. We combined reverse transcription and polymerase chain reaction (RT-PCR) with microdissection of individual renal tubule segments. Relative quantitation of the resulting amplified cDNA utilized densitometry of autoradiograms from Southern blots probed with a specific 32P labeled probe. The largest signals for BK2 receptor PCR product were detected in the cortical collecting tubule (CCT) and the proximal straight tubule (PST). BK2 receptor mRNA expression was also detected in the glomerulus, inner medullary thin limb (IMTL) and in the distal tubule (DT). Small but detectable signals were founded in the medullary thick ascending limb (MTAL). This distribution is consistent with multiple sites of BK action to account for different renal effects such as hemodynamics, water and electrolytic balance regulations.

Cyclosporine A decreases kallikrein and BK2 mRNA expression in the rat renal cortex.

The effect of subcutaneous injection of cyclosporine (20 mg/kg/day for 3 days) on the expression of kallikrein (Kal) and bradykinin 2 receptor (BK2) mRNA in the rat renal cortex was examined. CsA decreased significantly Kal and BK2 mRNA expression in the kidney cortex. These results indicate that the kallikrein-kinin system may participate in the genesis or the aggravation of the renal haemodynamic effect induced by long term administration of CsA.

Postnatal maturation of the Kallikrein-kinin system in the rat kidney: from enzyme activity to receptor gene expression.

During the course of aging, the balance between intrarenal hormones is disturbed. These age-related changes are well documented for the vasoconstrictor renin-angiotensin system, but comparable information on the renal kallikrein-kinin system is not yet available. The status of the kallikrein-kinin system was assessed by (1) kallikrein activity, measured by RIA; (2) maximum binding site density (Bmax) and affinity (Kd) of nonapeptide bradykinin (BK)-2 receptor, estimated by binding assays; (3) expression of BK2-receptor receptor mRNA, detected by reverse transcription-polymerase chain reaction (RT-PCR) using specific BK2-oligonucleotide primers. These parameters were determinated on renal glomeruli of 3-, 5-, 8-, 12- and 38-wk-old normotensive rats. Kallikrein activity increased from 3.2 to 7.7 ng BK/min per mg protein. The density of BK2 binding sites also rose from 12 to 40 femtomoles/mg protein with no difference in affinity. There was no change in specificity, which remained that expected of a BK2 receptor. The increase in the density of BK2 binding sites was associated with an augmented mRNA expression, whereas beta-actin mRNA used as a control remained unchanged. The ratio of BK2 mRNA to beta-actin mRNA indicated maximum steady expression after 8 wk of age. The data provide evidence that the renal kallikrein-kinin system develops postnatally.

Relationships between kallikrein secretion, kallikrein excretion and beta-adrenoceptors in kidney cortical slices from neurogenic hypertensive dogs.

1. Sinoaortic denervation (SAD) in dogs is characterized by an increase in blood pressure and heart rate as well as the development of renal morphological lesions similar to those observed in essential hypertension in human subjects. To assess the effect of SAD on the secretion of kallikrein kinin systems (KKS), we studied the in vitro secretion of kallikrein by renal cortical slices of normal and neurogenic hypertensive dogs (1 and 18 months after SAD). The method using renal cortical slices allowed the study of secretion of kallikrein independently of renal perfusion pressure. The number of renal beta-adrenoceptors was measured by [125I]-cyanopindolol binding. 2. SAD was associated with a marked increase in urinary kallikrein excretion at one month and a significant decrease at 18 months when compared with controls. Both changes were statistically significant (P < 0.05). Concurrently, a progressive increase in in vitro kallikrein secretion was observed (+80 +/- 10% and +179 +/- 48%, 1 and 18 months after SAD, respectively). Moreover, the cortical slices obtained from sinoaortic denervated dogs contained more kallikrein than the control cortical slices (+32 +/- 16% and +55 +/- 7%, 1 and 18 months after SAD, respectively). 3. Renal beta-adrenoceptor number significantly (P < 0.05) decreased 18 months after SAD from 18 +/- 2 to 8 +/- 3 fmol mg-1 protein without any change in affinity constant. 4. Although there was no test of association, because the number of renal beta-adrenoceptors decreased whereas kallikrein secretion increased, the present data could suggest a beta-adrenoceptor-mediated inhibition of kallikrein secretion. These results show that although the urinary kallikrein is decreased, the tissue secretory capacities are enhanced. This could suggest a renal compensatory mechanism possibly involved in tissue protection in dogs after SAD, although such a mechanism is not sufficient to reverse hypertension.

Comparison of B1 and B2 receptor activation on intracellular calcium, cell proliferation, and extracellular collagen secretion in mesangial cells from normal and diabetic rats.

The mesangial cell is a contractile secreting cell found in a key position in the renal glomerulus. Several kidney and systemic diseases are associated with dysfunctions of the mesangial cells. We compared the effect of bradykinin (BK; B2 agonist) and des-Arg9-bradykinin (DBK; B1 agonist) on intracellular calcium mobilization, cell proliferation, and collagen secretion of mesangial cells from normal and streptozotocin-induced diabetic rats. Stimulation of mesangial cells with BK and DBK caused an increase in intracellular calcium (Ca2+). However, the patterns of the Ca2+ increases induced by BK and DBK were different, indicating that DBK induced a major Ca2+ influx, whereas BK preferentially released Ca2+ from intracellular pools. Stimulation with BK and DBK did not show any heterologous desensitization, thus indicating the presence of two distinct binding sites. In normal cells, DBK stimulated cell proliferation more than BK, and this action was potentiated by insulin. No effect of BK or DBK was found in cells harvested from diabetic rats. The proliferation effect of BK and DBK was restored by insulin. DBK stimulated more collagen synthesis than BK in normal cells. In cells harvested from diabetic rats the collagen secretion was increased, but BK and DBK no longer had any effect. Insulin reduced basal collagen secretion in normal cells and cells harvested from diabetic rats. Insulin also blunted stimulation by BK and DBK in normal cells but did not restore the response to BK and DBK in cells harvested from diabetic rats. Our results show that the sensitivity to DBK and BK decreases during the course of insulin-dependent diabetes, indicating modulation by insulin.

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.

Bradykinin-induced in vitro contraction of rat mesangial cells via a B2 receptor type.

The effect of bradykinin (BK) on the contraction of rat mesangial cells (MC) was compared with that of various vasoactive agents. BK induced a dose-dependent contraction [one-half maximal effective dose (ED50) = 50 nM] inhibited by the B2 antagonist, HOE-140 (ED50 = 10 nM). BK-induced MC contraction was independent of extracellular calcium and was reduced by inhibition of protein kinase C (PKC). Neomycin completely prevented the increase in intracellular calcium and the formation of inositol 1,4,5-trisphosphate induced by BK but only reduced cell contraction. Inhibition of prostaglandin (PG) formation and administration of the endoperoxide antagonist SQ-27427 also partly decreased the effect of BK. Interestingly, only the addition of both neomycin and mepacrine resulted in a complete inhibition of cell contraction. These results suggest that BK, via a B2-kinin receptor, induces contraction of MC through two distinct mechanisms, one associated to the phospholipase C pathway and subsequent activation of PKC and the second one dependent on PG formation. These in vitro effects may be relevant in explaining the effects of BK and converting enzyme inhibitors on glomerular hemodynamics.

Renal immunolocalization of kallikrein in cisplatin nephrotoxicity in rats.

Treatment of rats with cisplatin (4 mg kg-1 body wt i.p. injection) induced variations of urinary kallikrein excretion (UKE). Three phases were observed: a transient increase of UKE one day after injection, followed by a decrease up to 10 days suggesting an altered biosynthesis and a recovery phase with return to normal control values, 21 days after injection. Early morphological lesions were observed in proximal tubule cells on day 1; severe changes and tubular necrosis were observed in the following days. Less marked changes were also present in distal tubules but the vacuolated and desquamated cells appeared in the lumen of the tubules. By immunocytochemical methods, kallikrein was observed in connecting tubule cells, but also in some proximal tubule cells and along the endothelial side of the glomerular basement membrane and urinary space of glomeruli. An intense labelling was present in desquamated epithelial cells in dilated lumen of tubules. This study provides evidence of the presence of immunoreactive kallikrein in the glomerulus, already reported during acute failure, and confirms the use of urinary kallikrein measurements as a useful non-invasive index to assess a possible nephrotoxic effect at the distal level.

Immunolocalization of renal kallikrein-like substance in rat urinary bladder.

The renal origin of kallikrein is now clearly established. However, the presence of kallikrein in urine raises questions about a possible physiological role of this enzyme at the urinary level. We have already demonstrated the presence of kallikrein-like substance in rat ureter. For establishing the continuity of the presence of kallikrein-like substance along the urinary tract we have studied the localization of immunoreactive kallikrein-like substance in urinary bladder of the normal rat by immunohistochemical methods for light- and electron-microscopy, using an antibody against rat urinary kallikrein. By light microscopy, kallikrein-like substance was found to be associated with the lamina propria, which is the connective tissue component which constitutes one layer of the bladder wall. Weak staining was present in the smooth-muscle layer. By immuno-electron microscopy, kallikrein-like substance was localized in fibroblasts which were present in the connective tissue and which penetrated into the layer of smooth muscle; immunoreactivity was observed in endoplasmic reticulum, Golgi apparatus and free polyribosomes. Immunolabelling was demonstrated in no other part of the wall bladder and in no other cellular component. The continuity of the presence of kallikrein-like substance from the kidney to the urinary bladder gives new indications concerning the significance of this system in renal physiology.

Increase in renal and urinary low and high molecular weight kininogens during chromate-induced acute renal failure in the rat: evidence for renal kininogen production.

In the present study, we investigated the plasma, urinary and intrarenal concentrations of low and high molecular weight kininogens during sodium chromate (25 mg/kg body weight)-induced acute renal failure (ARF) in the rat. Urinary kininogen underwent a transient increase with a maximum on day 7 (78 +/- 22 versus 4.2 +/- 1.6 ng bradykinin/mg creatinine) whereas plasma kininogen did not and glomerular filtration rate decreased (92 +/- 8 versus 895 +/- 70 microliters/min). The tissue level of kininogen was enhanced both in the cortex (1,319 +/- 123 versus 86 +/- 8 pg bradykinin Eq/mg protein) and in the medulla (1,673 +/- 138 versus 44 +/- 9 pg bradykinin Eq/mg protein) but more in the medulla (36 +/- 4- versus 15 +/- 3-fold). As plasma kininogen level was unchanged and glomerular filtration rate decreased, the increase in both renal concentration and urinary excretion of kininogen probably reflects stimulated renal production of kininogen in this model of ARF. Whether the evoked renal production of kininogen results from a local inflammatory response only or may subserve another physiological purpose remains to be elucidated.

Protective effect of cicletanine on hypertension-induced decreases in the renal kallikrein-kinin and prostaglandin systems in stroke-prone spontaneously hypertensive rats.

We investigated the effect of two oral (p.o.) doses of cicletanine (5 and 30 mg/kg/day) for 4 weeks on urinary excretion (UKE), renal concentration (RKC) of kallikrein, and prostaglandin E2 (PGE2) and 6-keto-PGF1 alpha urinary excretion of stroke-prone (SP) spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) rats submitted to a high sodium intake (1%). Both doses of cicletanine induced a significant antihypertensive effect in treated SHR as compared with hypertensive untreated controls (HC). After 4-week treatment, a significant difference in mortality was observed between normotensive controls (NC) (0%) and HC (84%). Both doses of cicletanine reduced the mortality of hypertensive animals (8% SHR with 5 mg and 24% SHR with 30 mg vs. 84% in HC). Whereas UKE and RKC were decreased in HC during the progression of untreated hypertension from week 1 to week 4, both doses of cicletanine administration significantly prevented this decrease. Consistently with maintenance of UKE during the course of hypertension, the level of tissue kallikrein was higher in hypertensive cicletanine-treated than in untreated SHR. This increased RKC was associated with a significantly higher rate of kallikrein biosynthesis. The increased level of the urinary excretion and tissue concentration of PGE2 and 6-keto-PGF1 alpha in cicletanine-treated SHR as compared with untreated animals was also of interest. This protective effect on PG excretion correlated with that on kallikrein excretion. The results confirm the efficiency of cicletatine as an antihypertensive treatment. The antihypertensive action includes protective effects on potential vasodepressor kallikrein-kinin and prostaglandin systems.(ABSTRACT TRUNCATED AT 250 WORDS)

Is yohimbine-induced increase in salivary secretion a kinin-dependent mechanism?

We have previously reported that the alpha 2-adrenoceptor antagonist yohimbine induced a significant increase in both salivary flow rate and kallikrein output. In order to assess the possible role of the kinin-kallikrein system in the increase in salivary secretion elicited by yohimbine, the effects of aprotinin, an inhibitor of kallikrein activity, were investigated in yohimbine-treated conscious dogs. Aprotinin (at a dose, 5000 IU/kg iv, which reduced both resting and yohimbine-induced increase in kininogenase and amidolytic activities of saliva) which remained inactive alone, failed to modify the increase in salivary volume elicited by yohimbine (0.5 mg/kg iv). These results show that the rise in salivary flow rate observed under alpha 2-adrenoceptor antagonist is not induced by the kinin-kallikrein system. The release of kallikrein into saliva observed after yohimbine is rather the consequence than the cause of the increase in salivary secretion.

Glomerular B2-kinin-binding sites in two-kidney, one-clip hypertensive rats.

To extend our recent observations of the possible downregulation of glomerular B2-kinin-binding sites, we investigated density (Bmax) of bradykinin (BK)-binding sites in glomerular membranes of both the clipped (C) and nonclipped (NC) kidneys of two-kidney, one-clip (2K-1C) Goldblatt hypertensive rats, in relation to tissue kallikrein activity and glomerular three-dimensional structure. Compared with the Bmax of sham-operated (SO) kidney (31.8 +/- 7 fmol/mg protein), a significant increase in Bmax was observed in glomeruli of both kidneys in hypertensive rats, the Bmax being higher in glomeruli of NC than in C kidneys (98 +/- 11 vs. 59 +/- 12 fmol/mg protein). NC kidney compensatory hypertrophy was expressed by an increase in glomerular diameter, surface area, and volume. When expressed per unit of area or volume, Bmax in NC kidneys remained significantly higher than in both C and SO kidneys. Increased Bmax in both kidneys of 2K-1C rats was associated with a decreased intrarenal level of kallikrein. We also examined prostaglandin (PG) E2 release by isolated glomeruli from SO, C, and NC kidneys as a possible biological effect induced by BK. Whereas C kidney released more PGE2 than NC kidney under basal conditions, addition of BK (10 nM) induced greater PGE2 production in NC kidney consistent with the difference in Bmax between C and NC kidneys. These results suggest a possible downregulation of glomerular B2-binding sites by bradykinin, which may explain the difference between SO and C kidneys.(ABSTRACT TRUNCATED AT 250 WORDS)

Cisplatin nephrotoxicity in cadmium-pretreated rats. Enzymatic, functional and morphological studies.

Treatment of rats with cisplatin or with cisplatin after chronic pre-exposure to cadmium induced a decrease in kidney cytochrome P-450 and glutathione levels, and in glutathione peroxidase and reductase activities. Furthermore, cadmium and cisplatin enhanced lipid peroxidation, oxidized glutathione and N-glucuronyl transferase activity. Glutathione S-transferase (substrate: 1-chloro-2,4-dinitrobenzene) was increased and decreased by cadmium and cisplatin respectively. On morphological observation, cadmium nephrotoxicity was characterized by tubular proximal damage with mitochondrial and lysosomal changes and a widespread vesiculation of tubular cells. A marked focal tubular necrosis associated with cyst formation was observed in cisplatin nephrotoxicity. On the basis of the measured biochemical, functional and histological parameters, it is concluded that cadmium pretreatment did not potentiate the nephrotoxic effect of cisplatin.

Distal nephrotoxicity of cisplatin demonstrated by urinary kallikrein excretion and morphological study in rats.

The nephrotoxic effect of cisplatin (4 mg/kg body wt, i.p. injection) was specifically evaluated on the distal tubule. We measured both the tissue concentration and the urinary excretion of kallikrein (UKE), a serine protease mainly synthesized and secreted in the distal connecting tubular cells. In a parallel morphological study, we evaluated the tissue lesions. On the basis of UKE, the three distinct phases of nephrotoxicity were observed. The induction phase, 1 day after cisplatin injection, was associated with a transient increase in UKE. During the maintenance phase, the kallikrein concentration was significantly decreased both in renal cortex and urine for up to 10 days, suggesting an alteration in the biosynthesis with a decrease in the activation of inactive kallikrein. The recovery phase, 21 days after cisplatin injection, was suggested by the incomplete but significant tendency to return towards control values of active UKE. Histological examinations of cisplatin-treated rats showed early lesions of proximal tubules on day 1. The injuries worsened and tubular necrosis was frequently observed on the following days. Distal tubular changes were less marked but vacuolization and desquamation of epithelial cells and swollen and disrupted mitochondria were demonstrated. This study adds new evidence that UKE is a useful and reliable non-invasive index to assess possible nephrotoxic effects in the distal tubule which are also directly visualized by histological lesions.

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.

Cisplatin-induced changes on cytochrome P-450, lipid peroxidation and some P-450 related specific catalytic activities in rat liver.

This study investigates the effects of three successive cisplatin administrations to rats on liver cytochrome P-450 and some related specific catalytic activities. Additionally, because glutathione and its related enzymatic system are significantly involved in cellular detoxification process, we examined the effects of cisplatin on lipid peroxidation, glutathione levels, glutathione reductase and peroxidase activities. Cisplatin induced a decrease of cytochrome P-450, glutathione-S-transferase and some cytochrome P-450 related specific catalytic activities among which aminopyrine demethylase and benzo(a)pyrene hydroxylase activities are representative of cytochrome P-450b and P-450h isoforms. Furthermore, cisplatin-induced N-glucuronyl transferase and lipid peroxidation which might participate, at least in part, in cisplatin-induced hepatotoxicity. The ability of cisplatin to cause alterations in the activity of other enzymes than those of cytochrome P-450 system suggests that cisplatin induces toxic effects in an unspecific manner.

Mild renal failure induced by subchronic exposure to molybdenum: urinary kallikrein excretion as a marker of distal tubular effect.

In this study we investigated the effect of two molybdenum (Mo) doses (40 and 80 mg/kg/d) on renal function. Neither dose of Mo was able to induce significant hypertension in treated animals. Subchronic exposure to high doses of Mo resulted in a delay in body weight gain associated with mild renal failure marked by a decrease in glomerular filtration. An increase in diuresis and urinary kallikrein excretion associated with unchanged glycosuria and proximal tubular enzymuria (alanine aminopeptidase and gamma-glutamyl transpeptidase) evoked a preferential mild effect at the distal tubule.