Signal transduction pathways involved in kinin B(2) receptor-mediated vasodilation in the rat isolated perfused kidney

The signal transduction pathways involved in kinin B(2) receptor-related vasodilation were investigated in rat isolated perfused kidneys. During prostaglandin F(2alpha) or KCl-induced constriction, the vasodilator response to a selective B(2) receptor agonist, Tyr(Me)(8)bradykinin (Tyr(Me)(8)BK), was assessed. Tyr(Me)(8)BK produced a concentration- and endothelium-dependent relaxation that was decreased by about 30 - 40% after inhibition of nitric oxide (NO) synthase by N(G)-nitro-L-arginine (L-NOARG) or of cyclo-oxygenase by indomethacin; a greater decrease (about 40 - 50%) was observed after concomitant inhibition of the two pathways. High extracellular K(+) diminished Tyr(Me)(8)BK-induced relaxation by about 75% suggesting a major contribution of endothelium-derived hyperpolarization. The residual response was almost completely suppressed by NO synthase and cyclo-oxygenase inhibition. The K(+) channel inhibitors, tetrabutylammonium (non-specific) and charybdotoxin (specific for Ca(2+)-activated K(+) channel), suppressed Tyr(Me)(8)BK-induced relaxation resistant to L-NOARG and indomethacin. Inhibition of cytochrome P450 (clotrimazole or 7-ethoxyresorufin) decreased the NO/prostanoids-independent relaxation to Tyr(Me)(8)BK by more than 60%, while inhibition of the cannabinoid CB(1) receptor (SR 141716A) had only a moderate effect. Acetylcholine induced a concentration-dependent relaxation with characteristics nearly similar to the response to Tyr(Me)(8)BK. In contrast, the relaxation elicited by sodium nitroprusside was potentiated in the absence of NO (L-NOARG or removal of endothelium) but remained unchanged otherwise. These results indicate that the activation of kinin B(2) receptors in the rat isolated kidney elicits an endothelium-dependent vasorelaxation, mainly dependent on the activation of charybdotoxin-sensitive Ca(2+)-activated K(+) channels. In addition, cytochrome P450 derivatives appear to be involved.     

Enalaprilat-mediated activation of kinin b(1) receptors and vasodilation in the rat isolated perfused kidney

The present study evaluated whether enalaprilat (the active form of enalapril, an angiotensin-converting enzyme inhibitor) activates B(1) receptors. We observed that the levels of B(1) receptor mRNA and protein expression were upregulated in the kidneys of diabetic rats. Bradykinin (BK)-induced renal vasodilation decreased in isolated perfused kidneys of diabetic rats, but des-Arg(9)-BK-induced renal vasodilation increased. Enalaprilat also produced vasodilation in the isolated perfused kidneys of control and diabetic rats. The response to des-Arg(9)-BK or enalaprilat was blocked by Lys-(des-Arg(9), Leu(8))-BK (a B(1) receptor antagonist) and N-nitro-L-arginine methyl ester (an inhibitor of nitric oxide synthase). These results suggest that enalaprilat activates B(1) receptors and stimulates the production of nitric oxide in the kidneys of both control and diabetic rats.     

Vascular catabolism of bradykinin in the isolated perfused rat kidney

Kinins in the circulation are rapidly metabolized by several different peptidases. The purpose of this study was to evaluate the contribution of membrane-bound peptidases to kinin metabolism in the renal circulation. Experiments were performed in vitro, in isolated rat kidneys perfused at a constant flow rate (8 ml/min) with Tyrode's solution. The effects of peptidase inhibitors were evaluated on the functional vasodilator response caused by bradykinin (30 nM) or [Tyr(Me)(8)]bradykinin (10 nM) via activation of bradykinin B2 receptors in kidneys precontracted with prostaglandin F2alpha. Angiotensin converting enzyme inhibitors, enalaprilat (3 microM), ramiprilat (1 microM) or lisinopril (1 microM), increased the bradykinin-induced renal vasodilation by 40% or more. Inhibitors of neutral endopeptidase (thiorphan or phosphoramidon, 10 microM), basic carboxypeptidase (DL-2-mercaptomethyl-3-guanidino-ethylthiopropanoic acid or MGTPA, 10 microM) and aminopeptidase P (apstatin, 20 microM) however did not enhance the renal vasodilator response elicited by kinins, whatever tested alone or in the presence of lisinopril. These findings indicate that angiotensin converting enzyme is the major peptidase whose inhibition potentiates the renal bradykinin B2 receptor mediated vasodilator response of kinins. The relative contribution in this potentiation of inhibition of kinin inactivation and of cross-talk of angiotensin converting enzyme with bradykinin B2 receptor remains however to be clarified.     

Vascular alpha1-adrenoceptors in isolated perfused rat kidney: influence of ageing

1. The present study identifies alpha1-adrenoceptor subtype(s) involved in constrictor responses of the kidney and how ageing influences it. 2. The study was conducted on kidneys from F344BNF1 rats, which unlike F344 or Wistar rats used by many previous investigators do not exhibit glomerulonephritis at advanced age. 3. Noradrenaline (NA) and phenylephrine (PHE) (non-selective alpha1) and A61063 (selective alpha(1A)) adrenoceptor agonists elicited constriction of perfused kidneys of young and old rats. The pD2 values (index of renovascular reactivity) were significantly higher for A61603 than for either PHE or NA, and significantly decrease across age groups. 4. BMY 7378 or RS 100329, alpha(1D)- or alpha(1A)-adrenoceptor antagonists, respectively antagonized the constrictor responses and suppressed the maximal responses to all agonists in young adult rat kidneys. However, antagonism of PHE or A61063 by BMY 7378 in old rat kidneys was surmountable. 5. This study suggests that: (i) alpha(1A) and alpha(1D)-adrenoceptor subtypes mediate vasoconstriction of perfused rat kidney; (ii) alpha(1A)-adrenoceptor subtype appears to predominate in renal vasculature based on agonist relative potencies. (iii) Ageing significantly decreases alpha1-adrenoceptor-mediated vasoconstriction of rat kidney.     

Tubular effects of acetaminophen in the isolated perfused rat kidney

 The effects of different acetaminophen (APAP) concentrations (1, 5 or 10 mM) on renal function were investigated in the isolated perfused rat kidney (IPK). APAP was added to the perfusion media as a single dose after a equilibration time and control periods. Changes in fractional excretion of sodium (FE_Na), water (FE_H2O), glucose (FE_glu) and in glomerular filtration rate (GFR) were measured. The lower concentration used only modified the FE_H2O. APAP 10 mM induced an increment in FE_H2O (72% higher than control preparation), FE_Na (79% higher than control preparation) and an elevation in glucose excretion (55% higher than control preparation), associated with a decrease in GFR (23% lower than control preparation). The influence of PGE₂ on the effects of APAP was also investigated. PGE₂ prevented the APAP-induced decrease in GFR and in glucose reabsorption, but did not change the pattern of sodium and water handling. The effects of another vasodilator, verapamil, on APAP-induced renal effects were also tested. Verapamil prevented the glomerular but not the tubular effects of APAP. Urinary APAP excretion data showed a similar availability of APAP to the tubular cells in all the groups. Our data suggest that APAP exerts a direct action in the IPK, affecting hemodynamic and tubular functions, and that the latter are not a consequence of hemodynamic alterations. Received: 30 May 1994/Accepted: 19 August 1994     

Vascular effects of apomorphine and related compounds in the perfused rat kidney

The renal vascular effects of aporphines and related compounds were studied on the isolated perfused rat kidney in the presence of 10(-5) M phenoxybenzamine and 10(-5) M sotalol and after contraction of the vascular bed with prostaglandin F2 alpha (10(-7) -3 X 10(-6) M). Under these conditions, (R)-(-)-apomorphine showed renal dopaminomimetic activity, i.e. renal vasodilation competitively inhibited by (+)-butaclamol (10(-8) M) but not by (-)-butaclamol (3 X 10(-8) M). It had an apparent affinity 25 times higher but a markedly lower intrinsic activity than dopamine. N-n-Propyl and trihydroxylated aporphines were less potent and the mono-10-hydroxylated aporphine was completely inactive. (S)-(+)-Bulbocapnine also showed weak dopaminomimetic activity but tetrahydropapaveroline was devoid of such an effect. (-)-N-(2-Chloroethyl)-norapomorphine (10(-5) M) irreversibly antagonised dopamine-induced renal vasodilation. At concentrations above 3 X 10(-6) M, most aporphines and tetrahydropapaveroline induced additional non-dopamine receptor related renal vasodilation.     

Vascular effects of capsaicin in isolated perfused rat mesenteric bed

The effects of intra- and extraluminal capsaicin administration were evaluated in isolated perfused rat mesenteric bed. Capsaicin (10 nM-1 microM) produced a potent concentration-dependent relaxation of the tonic vasoconstriction induced by norepinephrine (1 microM) but not by high-K+ (60 mM). The capsaicin-induced relaxation was nearly abolished in preparations pretreated in vitro with a high concentration of capsaicin (1 microM, for 10 min, 1 h before). Capsaicin-induced relaxation was reduced but not abolished in preparations obtained from rats pretreated neonatally with capsaicin. The capsaicin effects were unaffected by atropine, guanethidine, propranolol, hexamethonium or tetrodotoxin. The observation that capsaicin (0.1 microM)-induced relaxation was virtually abolished in presence of the proteolytic enzyme alpha-chymotrypsin (1 U/ml) supports the involvement of neuropeptide(s) in this response. Bolus injections of calcitonin gene-related peptide (CGRP) elicited a potent and rapidly ensuing relaxation which underwent tachyphylaxis. However, no cross-desensitization with capsaicin was observed. It is concluded that activation of capsaicin-sensitive sensory fibers could release neuropeptides locally with a potent effect on intestinal blood flow.     

Renal handling and effects of S(+)-ibuprofen and R(-)-ibuprofen in the rat isolated perfused kidney.

1. The renal handling and effects of S(+)- and R(-)-ibuprofen have been studied in the isolated perfused kidney (IPK) of the rat. 2. Both ibuprofen enantiomers were extensively reabsorbed and accumulated in the kidney in a concentration-dependent manner. No pharmacokinetic differences were observed between the two enantiomers. 3. S(+)-ibuprofen concentrations ranging from 0.25 to 25 micrograms ml-1 (1.2 to 120 microM) caused a decrease in urinary flow, glomerular filtration rate (GFR) and electrolyte excretion. Urinary pH and excretion of glucose were not influenced. R(-)-ibuprofen concentrations ranging from 2.5 to 25 micrograms ml-1 (12 to 120 microM) also decreased urinary flow and electrolyte excretion. This decrease, however, was less than observed with S(+)-ibuprofen. GFR, urinary pH and glucose excretion were not affected by R(-)-ibuprofen. Prostaglandin E2 (PGE2) concentrations of 133 ng ml-1 reversed the effects on renal function of both enantiomers. 4. Very high S(+)- and R(-)-ibuprofen concentrations (greater than 400 micrograms ml-1) resulted in an increase in urinary flow and fractional excretion of sodium, chloride, potassium, glucose and calcium. 5. It is concluded that the pharmacokinetic behaviour of ibuprofen in the kidney is not stereoselective. Relatively high concentrations of both enantiomers increased the urinary flow and electrolyte excretion in a nonstereoselective manner. Lower concentrations of S(+)-ibuprofen decreased urinary flow and electrolyte excretion. The pharmacologically inactive R(-)-ibuprofen was also able to affect renal function in a similar way, but at different concentrations. These effects on renal function are probably caused by inhibition of PGE2 synthesis.     

Indometacin: renal handling and effects in the isolated perfused rat kidney

We investigated renal handling and effects of indometacin on renal function in the isolated perfused rat kidney (IPK). Indometacin concentrations less than 2.5 ng/ml did not influence renal function, while higher concentrations caused a decrease in urinary flow and electrolyte excretion. The presence of 133 ng/ml prostaglandin E2 (PGE2) in the perfusate fully opposed these effects on kidney function. Only a small fraction of the filtered indometacin is excreted into the urine, indicating extensive reabsorption of the compound. This is probably a passive process, dependent on the tubular load, urinary pH and urinary flow. Indometacin accumulates extensively in the IPK, causing a kidney to perfusate ratio between 5 and 9. Accumulation decreased with increasing perfusate concentration. This can be explained by active secretion: increasing the perfusate concentration leads to a saturation of the active secretion and a decrease in the relative accumulation. We conclude that indometacin accumulates extensively in the IPK, that it affects kidney function and that this influence is probably caused by the inhibition of PGE2 synthesis.     

Vascular resistance in the perfused isolated rat tail

1. A constant flow perfusion system using the isolated rat tail has been developed to facilitate the study of resistance vessel behaviour and the action of vasoactive drugs.2. Baseline resistance remains stable for several hours and dose response curves to bolus injections of pressor agents are reproducible when dialysed bovine serum albumen is used in the perfusion medium to maintain osmotic pressure.3. Noradrenaline, adrenaline, serotonin, vasopressin, angiotensin II, high potassium concentrations and sympathetic nerve stimulation constricted the vascular bed.4. Angiotensin I, bradykinin, histamine, acetylcholine and isoprenaline did not alter vascular resistance under baseline conditions.5. Maximal sensitivity to noradrenaline occurred at 32 degrees to 34 degrees C. Below 30 degrees C, resting tone increased and the pressor effect of noradrenaline was prolonged.6. Low concentrations of (+/-)-propranolol in the perfusate enhanced adrenaline and noradrenaline vasoconstriction, high concentration of (+/-)-propranolol had a direct pressor effect and did not affect catecholamine responses.7. The preparation is a simple and relatively inexpensive adjunct to established methods of studying resistance vessel behaviour under varying experimental conditions.     

Renal handling and effects of salicylic acid in the isolated perfused rat kidney

The renal handling of salicylic acid (SA) and its effects on renal function were studied in the isolated perfused rat kidney (IPK). The renal handling of SA is dominated by reabsorption and only a small fraction of the filtered SA is excreted into the urine. Reabsorption is a passive process and is dependent on urinary pH. Because of the extensive reabsorption, no decrease in perfusate concentration can be observed in the course of the IPK experiment. SA accumulated slightly in the IPK and this accumulation is concentration dependent. Small amounts of SA were converted to salicyluric acid (SU), the glycine conjugate of SA. SA concentrations higher than 100 micrograms/ml caused an immediate increase in urinary flow and in fractional excretion of sodium, potassium, chloride and calcium. Fractional excretion of glucose increased gradually. Glomerular filtration rate, renal perfusion flow, renal pressure and fractional excretion of magnesium were not affected by SA. The effects were dependent on the SA concentration. Although SA is a classical non-steroidal antiinflammatory drug (NSAID), its influence on renal function appears to be different from other NSAIDs which are usually associated with a reduction in urinary flow and salt excretion.     

Hemodynamic and tubular effects of endothelin and thromboxane in the isolated perfused rat kidney

We have investigated the effects of the endothelium-derived peptide, endothelin, and of a chemically stable analog of thromboxane (TX) A2, U46619, on the glomerular hemodynamics and tubular function of isolated, perfused rat kidneys. Endothelin (10(-11)-10(-9) M) dose dependently decreased the renal plasma flow to a significantly greater extent than it did the glomerular filtration rate. In contrast, U46619 (10(-9)-10(-7) M) had more pronounced effects on the glomerular filtration rate than on the renal plasma flow. As a consequence, the filtration fraction was increased by endothelin and decreased by U46619. Endothelin, unlike U46619, enhanced urinary Na+ excretion and reduced oxygen consumption at concentrations that greatly decreased the tubular load, thus suggesting that it has a direct effect on tubular Na+ reabsorption. Addition of the TXA2/PGH2-receptor antagonist, BM13177 (4.10(-4) M), or the cyclooxygenase inhibitor, acetylsalicylic acid (10(-3) M), to the perfusion medium failed to modify the endothelin-induced increase in Na+ excretion or the reduction in renal plasma flow, whereas acetylsalicylic acid, but not BM13177, partially prevented the decrease in the glomerular filtration rate. These results demonstrate that two contractile agonists produced by the kidney have specific and differential effects on cortical and tubular functions. Moreover, the intrarenal production of eicosanoids does not appear to play a major role in the renal effects of endothelin.     

Biotransformation of the hexachlorobutadiene metabolites 1-(glutathion-S-yl)-pentachlorobutadiene and 1-(cystein-S-yl)-pentachlorobutadiene in the isolated perfused rat liver

1. The first step in the bioactivation of the nephrotoxin hexachlorobutadiene is the biosynthesis of 1-(glutathion-S-yl)-1,2,3,4,4-pentachloro-1,3-butadiene (GPCB). GPCB formed in the liver is secreted into bile, may be reabsorbed in the gut, intact or after hydrolysis to 1-(cystein-S-yl)-1,2,3,4,4-pentachloro-1,3-butadiene (CPCB), and undergo enterohepatic circulation or translocation to the kidney. Hepatic uptake and metabolism of GPCB and CPCB may thus influence the disposition of these S-conjugates. We therefore studied the metabolism and uptake of CPCB and GPCB in the isolated perfused rat liver.

2. Dose-dependent uptake of GPCB and CPCB from the perfusion medium by isolated perfused liver was demonstrated; CPCB is cleared from the perfusion medium to a much higher extent than GPCB.

3. GPCB and CPCB are intensively biotransformed to biliary metabolites. These metabolites were identified by thermospray mass spectrometry as products of the conjugation reaction of GPCB and CPCB with glutathione and subsequent hydrolysis of the glutathione moieties.

4. Hepatic biosynthesis of 1-(N-acetyl-L-cystein-S-yl)-1,2,3,4,4-pentachloro-1,3-butadiene from CPCB was only a very minor pathway in GPCB and CPCB metabolism in liver.

5. The results indicate that hepatic biosynthesis of mercapturic acids may not contribute to the disposition of S-conjuagates formed from hexachlorobutadiene in vivo and that GPCB may be, at least in part, delivered intact to the kidney.     

Flow-dependent extraction of 1-naphthol by the rat isolated perfused kidney

Summary The influence of variation of perfusion flow rate on the renal clearance of p-aminohippuric acid and 1-naphthol was studied with an isolated perfused rat kidney preparation. Kidney functions were well maintained at low perfusion flow rates by the use of a fluorocarbon emulsion to increase the oxygen capacity of the perfusion buffer. Renal extraction of p-aminohippuric acid decreased with increasing perfusion flow. Our data show that at high perfusion flow rates maximal extractable perfusion flow forms only a small part of the total perfusion flow. 1-Naphthol is rapidly metabolized to its glucuronide and sulfate conjugate in the isolated perfused rat kidney. Using PAH as a marker for the maximal extractable perfusion flow, 1-naphthol could be regarded as a high-extraction compound even at high perfusion flow rates. Our results suggest that p-aminohippuric acid clearance, rather than total perfusion flow rate, should be used as the measure of maximal extractable blood flow for the estimation of extraction ratio in the isolated perfused kidney of compounds excreted or metabolized by the proximal tubules.     

Prednisolone metabolism and excretion in the isolated perfused rat kidney

The isolated perfused rat kidney was used to identify factors responsible for the renal elimination of prednisolone (Pn). Pn was recirculated at initial concentrations varying from 100 to 1000 ng/ml for 90 min. Perfusate and urine samples were assayed for Pn and prednisone by HPLC. Protein binding of Pn was measured by using 3H-Pn and equilibrium dialysis at 37 degrees C. There were no significant differences in perfusate flow, glomerular filtration rate, urine flow, or sodium excretion between control and steroid experiments. Partial metabolism of Pn to prednisone occurred in all studies. The total kidney clearance (CIT) of Pn ranged from 0.39 to 1.24 ml/min/100 g of rat body weight with approximately half of the Pn dose unaccountable for as either Pn or prednisone. The apparent percentage of the Pn dose excreted unchanged in the urine ranged from 1.9 to 6.4% and was not related to Pn dose. The apparent urinary clearances of Pn and its metabolite, prednisone, normalized for inulin clearance (fractional excretion) were variable with means of 0.068 and 0.095, respectively. The fractional excretions of Pn and prednisone were related to the fraction of filtered water excreted but not to perfusate concentration. Thus, the extent of urinary clearance of these corticosteroids is related to glomerular filtration and passive tubular reabsorption. The perfused rat kidney reflects the urinary and renal metabolic clearance of Pn without the complication of dose-dependent disposition.     

The toxicokinetics of (1R, cis)- and (1R, trans)- tetramethrin in the isolated perfused rat liver

1. The toxicokinetics of cis- and trans-tetramethrin isomers were investigated using the isolated perfused rat liver preparation. 2. The concentration of cis- and trans-tetramethrin decreased rapidly in the plasma perfusate and was initially replaced by N-(hydroxymethyl)3,4,5,6-tetrahydrophthalimide (MTI) and then by 3,4,5,6-tetrahydrophthalimide (TPI). Plasma perfusate concentrations of the intact cis-isomer were higher than those of the trans-isomer. Concentrations of MTI and TPI were higher in livers treated with the trans-isomer. 3. Tetramethrin and its metabolites were rapidly excreted in the bile. Bile from livers perfused with trans-isomer contained higher concentrations of parent isomer and metabolites MTI and TPI, than did bile from livers treated with the cis-isomer.     

Differential effects of uridine adenosine tetraphosphate on purinoceptors in the rat isolated perfused kidney

BACKGROUND AND PURPOSE

Purinergic signalling plays an important role in vascular tone regulation in humans. We have identified uridine adenosine tetraphosphate (Up₄A) as a novel and highly potent endothelial-derived contracting factor. Up₄A induces strong vasoconstrictive effects in the renal vascular system mainly by P2X₁ receptor activation. However, other purinoceptors are also involved and were analysed here.

EXPERIMENTAL APPROACH

The rat isolated perfused kidney was used to characterize vasoactive actions of Up₄A.

KEY RESULTS

After desensitization of the P2X₁ receptor by α,β-methylene ATP (α,β-meATP), Up₄A showed dose-dependent P2Y₂-mediated vasoconstriction. Continuous perfusion with Up₄A evoked a biphasic vasoconstrictor effect: there was a strong and rapidly desensitizing vasoconstriction, inhibited by P2X₁ receptor desensitization. In addition, there is a long-lasting P2Y₂-mediated vasoconstriction. This vasoconstriction could be blocked by suramin, but not by PPADS or reactive blue 2. In preparations of the rat isolated perfused kidney model with an elevated vascular tone, bolus application of Up₄A showed a dose-dependent vasoconstriction that was followed by a dose-dependent vasodilation. The vasoconstriction was in part sensitive to P2X₁ receptor desensitization by α,β-meATP, and the remaining P2Y₂-mediated vasoconstriction was only inhibited by suramin. The Up₄A-induced vasodilation depended on activation of nitric oxide synthases, and was mediated by P2Y₁ and P2Y₂ receptor activation.

CONCLUSIONS AND IMPLICATIONS

Up₄A activated P2X₁ and P2Y₂ receptors to act as a vasoconstrictor, whereas endothelium-dependent vasodilation was induced by P2Y_1/2 receptor activation. Up₄A might be of relevance in the physiology and pathophysiology of vascular tone regulation.