Facilitation of renal autoregulation by angiotensin II is mediated through modulation of nitric oxide

Aims: This study was designed to investigate the influence of angiotensin II (Ang II) and nitric oxide (NO) on autoregulation of renal perfusion. Methods: Autoregulation was investigated in isolated perfused kidneys (IPRK) from Sprague-Dawley rats during stepped increases in perfusion pressure. Results: Ang II (75-200 pM) produced dose-dependent enhancement of autoregulation whereas phenylephrine produced no enhancement and impaired autoregulation of GFR. Enhancement by Ang II was inhibited by the AT(1) antagonist, Losartan, and the superoxide scavenger, Tempol. Under control conditions nitric oxide synthase (NOS) inhibition by 10 muM N-omega-nitro-L-arginine methyl ester (L-NAME) facilitated autoregulation in the presence of non-specific cyclooxygenase (COX) inhibition by 10 muM indomethacin. Both COX and combined NOS/COX inhibition reduced the autoregulatory threshold concentration of Ang II. Facilitation by 100 pM Ang II was inhibited by 100 muM frusemide. Methacholine (50 nM) antagonised Ang II-facilitated autoregulation in the presence and absence of NOS/COX inhibition. Infusion of the NO donor, 1 muM sodium nitroprusside, inhibited L-NAME enhancement of autoregulation under control conditions and during Ang II infusion. Conclusions: The results suggest than an excess of NO impairs autoregulation under control conditions in the IPRK and that endogenous and exogenous NO, vasodilatory prostaglandins and endothelium-derived hyperpolarizing factor (EDHF) activity antagonise Ang II-facilitated autoregulation. Ang II also produced a counterregulatory vasodilatory response that included prostaglandin and NO release. We suggest that Ang II facilitates autoregulation by a tubuloglomerular feedback-dependent mechanism through AT(1) receptor-mediated depletion of nitric oxide, probably by stimulating generation of superoxide.

Repetitive brief ischemia: Intermittent reperfusion during ischemia ameliorates the extent of injury in the perfused kidney

Acute renal failure commonly follows reduced renal perfusion or ischemia. Reperfusion is essential for recovery but can itself cause functional and structural injury to the kidney. The separate contributions of ischemia and of reperfusion were examined in the isolated perfused rat kidney. Three groups were studied: brief (5 min) ischemia, 20 min ischemia, and repetitive brief ischemia (4 periods of 5 min) with repetitive intervening reperfusion of 5 min. A control group had no intervention, the three ischemia groups were given a baseline perfusion of 30 min before intervention and all groups were perfused for a total of 80 min. In addition, the effects of exogenous (NO)-N-. from sodium nitroprusside and xanthine oxidase inhibition by allopurinol were assessed in the repetitive brief ischemia-reperfusion model. Brief ischemia produced minimal morphological injury with near normal functional recovery. Repetitive brief ischemia reperfusion caused less functional and morphological injury than an equivalent single period of ischemia (20 min) suggesting that intermittent reperfusion is less injurious than ischemia alone over the time course of study. Pretreatment with allopurinol improved renal function after repetitive brief ischemia-reperfusion compared with the allopurinol-untreated repetitive brief ischemia-reperfusion group. Similarly, sodium nitroprusside reduced renal vascular resistance but did not improve the glomerular filtration rate or sodium reabsorption in the repetitive brief ischemia-reperfusion model. Thus, these studies show that the duration of uninterrupted ischemia is more critical than reperfusion in determining the extent of renal ischemia-reperfusion injury and that allopurinol, in particular, counteracts the oxidative stress of reperfusion.

Angiotensin II facilitates autoregulation in the perfused mouse kidney: An optimized in vitro model for assessment of renal vascular and tubular function

Background and Aims: We have optimized the isolated perfused mouse kidney (IPMK) model for studying renal vascular and tubular function in vitro using 24-28 g C57BL6J mice; the wild type controls for many transgenic mice. Methods and Results: Buffer composition was optimized for bovine serum albumin concentration (BSA). The effect of adding erythrocytes on renal function and morphology was assessed. Autoregulation was investigated during stepped increases in perfusion pressure. Perfusion for 60 min at 90-110 mmHg with Krebs bicarbonate buffer containing 5.5% BSA, and amino acids produced functional parameters within the in vivo range. Erythrocytes increased renal vascular resistance (3.8 +/- 0.2 vs 2.4 +/- 0.1 mL/min.mmHg, P < 0.05), enhanced sodium reabsorption (FENa = 0.3 +/- 0.08 vs 1.5 +/- 0.7%, P < 0.05), produced equivalent glomerular filtration rates (GFR; 364 +/- 38 vs 400 +/- 9 muL/min per gkw) and reduced distal tubular cell injury in the inner stripe (5.8 +/- 1.7 vs 23.7 +/- 3.1%, P < 0.001) compared to cell free perfusion. The IPMK was responsive to vasoconstrictor (angiotensin II, EC50 100 pM) and vasodilator (methacholine, EC50 75 nM) mediators and showed partial autoregulation of perfusate flow under control conditions over 65-85 mmHg; autoregulatory index (ARI) of 0.66 +/- 0.11. Angiotensin II (100 pM) extended this range (to 65-120 mmHg) and enhanced efficiency (ARI 0.21 +/- 0.02, P < 0.05). Angiotensin II facilitation was antagonized by methacholine (ARI 0.76 +/- 0.08) and papaverine (ARI 0.91 +/- 0.13). Conclusion: The IPMK model is useful for studying renal physiology and pathophysiology without systemic neurohormonal influences.

Kinetic analysis of vascular marker distribution in perfused rat livers after regeneration following partial hepatectomy

Background/Aims: Liver clearance models are based on information (or assumptions) on solute distribution kinetics within the microvasculatory system, The aim was to study albumin distribution kinetics in regenerated livers and in livers of normal adult rats, Methods: A novel mathematical model was used to evaluate the distribution space and the transit time dispersion of albumin in livers following regeneration after a two-thirds hepatectomy compared to livers of normal adult rats. Outflow curves of albumin measured after bolus injection in single-pass perfused rat livers were analyzed by correcting for the influence of catheters and fitting a long-tailed function to the data. Results: The curves were well described by the proposed model. The distribution volume and the transit time dispersion of albumin observed in the partial hepatectomy group were not significantly different from livers of normal adult rats. Conclusions: These findings suggest that the distribution space and the transit time dispersion of albumin (CV2) is relatively constant irrespective of the presence of rapid and extensive repair. This invariance of CV2 implies, as a first approximation, a similar degree of intrasinusoidal mixing, The finding that a sum of two (instead of one) inverse Gaussian densities is an appropriate empirical function to describe the outflow curve of vascular indicators has consequences for an improved prediction of hepatic solute extraction.

Kinetics of propranolol uptake in muscle, skin, and fat of the isolated perfused rat hindlimb

The tissue distribution kinetics of a highly bound solute, propranolol, was investigated in a heterogeneous organ, the isolated perfused limb, using the impulse-response technique and destructive sampling. The propranolol concentration in muscle, skin, and fat as well as in outflow perfusate was measured up to 30 min after injection. The resulting data were analysed assuming (1) vascular, muscle, skin and fat compartments as well mixed (compartmental model) and (2) using a distributed-in-space model which accounts for the noninstantaneous intravascular mixing and tissue distribution processes but consists only of a vascular and extravascular phase (two-phase model). The compartmental model adequately described propranolol concentration-time data in the three tissue compartments and the outflow concentration-time curve (except of the early mixing phase). In contrast, the two-phase model better described the outflow concentration-time curve but is limited in accounting only for the distribution kinetics in the dominant tissue, the muscle. The two-phase model well described the time course of propranolol concentration in muscle tissue, with parameter estimates similar to those obtained with the compartmental model. The results suggest, first that the uptake kinetics of propranolol into skin and fat cannot be analysed on the basis of outflow data alone and, second that the assumption of well-mixed compartments is a valid approximation from a practical point of view las, e.g., in physiological based pharmacokinetic modelling). The steady-state distribution volumes of skin and fat were only 16 and 4%, respectively, of that of muscle tissue (16.7 ml), with higher partition coefficient in fat (6.36) than in skin (2.64) and muscle (2.79. (C) 2000 Elsevier Science B.V. All rights reserved.

Melphalan dosing regimens for management of recurrent melanoma by isolated limb perfusion: Application of a physiological pharmacokinetic model based on melphalan distribution in the isolated perfused rat hindlimb

The optimal dosing schedule for melphalan therapy of recurrent malignant melanoma in isolated limb perfusions has been examined using a physiological pharmacokinetic model with data from isolated rat hindlimb perfusions (IRHP), The study included a comparison of melphalan distribution in IRHP under hyperthermia and normothermia conditions. Rat hindlimbs were perfused with Krebs-Henseleit buffer containing 4.7% bovine serum albumin at 37 or 41.5 degrees C at a flow rate of 4 ml/min. Concentrations of melphalan in perfusate and tissues were determined by high performance liquid chromatography with fluorescence detection, The concentration of melphalan in perfusate and tissues was linearly related to the input concentration. The rate and amount of melphalan uptake into the different tissues was higher at 41.5 degrees C than at 37 degrees C. A physiological pharmacokinetic model was validated from the tissue and perfusate time course of melphalan after melphalan perfusion. Application of the model involved the amount of melphalan exposure in the muscle, skin and fat in a recirculation system was related to the method of melphalan administration: single bolus > divided bolus > infusion, The peak concentration of melphalan in the perfusate was also related to the method of administration in the same order, Infusing the total dose of melphalan over 20 min during a 60 min perfusion optimized the exposure of tissues to melphalan whilst minimizing the peak perfusate concentration of melphalan. It is suggested that this method of melphalan administration may be preferable to other methods in terms of optimizing the efficacy of melphalan whilst minimizing the limb toxicity associated with its use in isolated limb perfusion.

The effects of perfusion conditions on melphalan distribution in the isolated perfused rat hindlimb bearing a human melanoma xenograft

An isolated rat hindlimb perfusion model carrying xenografts of the human melanoma cell line MM96 was used to study the effects of perfusion conditions on melphalan distribution. Krebs-Henseleit buffer and Hartmann's solution containing 4.7% bovine serum albumin (BSA) or 2.8% dextran 40 were used as perfusates. Melphalan concentrations in perfusate, tumour nodules and normal tissues were measured using high-performance liquid chromatography (HPLC). Increasing the perfusion flow rates (from 4 to 8 mi min(-1)) resulted in higher tissue blood flow (determined with Cr-51-labelled microspheres) and melphalan uptake by tumour and normal tissues. me distribution of melphalan within tumour nodules and normal tissues was similar for both Krebs-Henseleit buffer and Hartmann's solution; however, tissue concentrations of melphalan were significantly higher for a perfusate containing 2.8% dextran 40 than for one containing 4.7% BSA. The melphalan concentration in the tumour was one-third of that found in the skin if the perfusate contained 4.7% BSA. In conclusion, this study has shown that a high perfusion flow enhances the delivery of melphalan into implanted tumour nodules and normal tissues, and a perfusate with low melphalan binding (no albumin) is preferred for maximum uptake of drug by the tumour.

Tissue and perfusate pharmacokinetics of melphalan in isolated perfused rat hindlimb

Melphalan is commonly used as a cytotoxic agent in isolated limb perfusion for locally recurrent malignant melanoma. The time course of melphalan concentrations in perfusate and tissues during a 60-min melphalan perfusion and 30-min drug-free washout in the single-pass perfused rat hindlimb was examined using a physiologically based pharmacokinetic model. The rat hindlimbs were perfused with Krebs-Heinseleit buffer containing 4.7% bovine serum albumin (BSA) or 2.8% dextran 40 at a constant rate of 3.8 ml/min. The concentration of melphalan in perfusate and tissues was determined by highperformance liquid chromatography. The tissue concentrations of melphalan were significantly higher with the perfusate containing dextran than BSA during the 60-min perfusion. During the washout period, the melphalan concentration in the perfusates decreased rapidly in first few minutes, followed by a slower monoexponential decline. The estimated half life (t(1/2)) for melphalan removal from skin and fat was 59 +/- 2 min for both BSA and dextran perfusates. However, the estimated t(1/2) for melphalan removal from muscle was 79 and 96 min for BSA and dextran washout perfusates, respectively. The predicted concentration-time profiles obtained for melphalan with BSA and dextran perfusates appear to correspond closely to the observed data. This study showed that the uptake of melphalan into perfused tissues is impaired by the use of perfusates in which melphalan is highly bound. Melphalan washout from muscle, but not skin and fat, was facilitated by the use of perfusates in which melphalan is highly protein bound.

Impulse-response studies on tracer doses of [C-14]lignocaine and its multiple metabolites in the perfused rat liver

The outflow-concentration-time profiles for lignocaine (lidocaine) and its metabolites have been measured after bolus impulse administration of [C-14]lignocaine into the perfused rat liver. Livers from female Sprague-Dawley rats were perfused in a once-through fashion with red-blood-cell-free Krebs-Henseleit buffer containing 0 or 2% bovine serum albumin. Perfusate flow rates of 20 and 30 mL min(-1) were used and both normal and retrograde flow directions were employed. Significant amounts of metabolite were detected in the effluent perfusate soon after lignocaine injection. The early appearance of metabolite contributed to bimodal outflow profiles observed for total C-14 radioactivity. The lignocaine outflow profiles were well characterized by the two-compartment dispersion model, with efflux rate << influx rate. The profiles for lignocaine metabolites were also characterized in terms of a simplified two-compartment dispersion model. Lignocaine was found to be extensively metabolized under the experimental conditions with the hepatic availability ranging between 0.09 and 0.18. Generally lignocaine and metabolite availability showed no significant change with alterations in perfusate flow rate from 20 to 30 mt min(-1) or protein content from 0 to 2%. A significant increase in lignocaine availability occurred when 1200 mu M unlabelled lignocaine was added to the perfusate. Solute mean transit times generally decreased with increasing flow rate and with increasing perfusate protein content. The results confirm that lignocaine pharmacokinetics in the liver closely follow the predictions of the well-stirred model. The increase in lignocaine availability when 1200 mu M unlabelled lignocaine was added to the perfusate is consistent with saturation of the hydroxylation metabolic pathways of lignocaine metabolism.

Structure-hepatic disposition relationships for cationic drugs in isolated perfused rat livers: Transmembrane exchange and cytoplasmic binding process

This work studied the structure-hepatic disposition relationships for cationic drugs of varying lipophilicity using a single-pass, in situ rat liver preparation. The lipophilicity among the cationic drugs studied in this work is in the following order: diltiazem. propranolol. labetalol. prazosin. antipyrine. atenolol. Parameters characterizing the hepatic distribution and elimination kinetics of the drugs were estimated using the multiple indicator dilution method. The kinetic model used to describe drug transport (the two-phase stochastic model) integrated cytoplasmic binding kinetics and belongs to the class of barrier-limited and space-distributed liver models. Hepatic extraction ratio (E) (0.30-0.92) increased with lipophilicity. The intracellular binding rate constant (k(on)) and the equilibrium amount ratios characterizing the slowly and rapidly equilibrating binding sites (K-S and K-R) increase with the lipophilicity of drug (k(on) : 0.05-0.35 s(-1); K-S : 0.61-16.67; K-R : 0.36-0.95), whereas the intracellular unbinding rate constant (k(off)) decreases with the lipophilicity of drug (0.081-0.021 s(-1)). The partition ratio of influx (k(in)) and efflux rate constant (k(out)), k(in)/k(out), increases with increasing pK(a) value of the drug [from 1.72 for antipyrine (pK(a) = 1.45) to 9.76 for propranolol (pK(a) = 9.45)], the differences in k(in/kout) for the different drugs mainly arising from ion trapping in the mitochondria and lysosomes. The value of intrinsic elimination clearance (CLint), permeation clearance (CLpT), and permeability-surface area product (PS) all increase with the lipophilicity of drug [CLint (ml . min(-1) . g(-1) of liver): 10.08-67.41; CLpT (ml . min(-1) . g(-1) of liver): 10.80-5.35; PS (ml . min(-1) . g(-1) of liver): 14.59-90.54]. It is concluded that cationic drug kinetics in the liver can be modeled using models that integrate the presence of cytoplasmic binding, a hepatocyte barrier, and a vascular transit density function.

Disposition of naproxen, naproxen acyl glucuronide and its rearrangement isomers in the isolated perfused rat liver

1. An isolated perfused rat liver (IPRL) preparation was used to investigate separately the disposition of the non-steroidal anti-inflammatory drug (NSAID) naproxen (NAP), its reactive acyl glucuronide metabolite (NAG) and a mixture of NAG rearrangement isomers (isoNAG), each at 30 mug NAP equivalents ml(-1) perfusate (n = 4 each group). 2. Following administration to the IPRL, NAP was eliminated slowly in a log-linear manner with an apparent elimination half-life (t(1/2)) of 13.4 +/-4.4 h. No metabolites were detected in perfusate, while NAG was the only metabolite present in bile in measurable amounts (3.9 +/-0.8%, of the dose). Following their administration to the IPRL, both NAG and isoNAG were rapidly hydrolysed (t(1/2) in perfusate=57 +/-3 and 75 +/- 14min respectively). NAG also rearranged to isoNAG in the perfusate. Both NAG and isoNAG were excreted intact in bile (24.6 and 14.8% of the NAG and isoNAG doses, respectively). 3. Covalent NAP-protein adducts in the liver increased as the dose changed from NAP to NAG to isoNAG (0.20 to 0.34 to 0.48% of the doses, respectively). Similarly, formation of covalent NAP-protein adducts in perfusate were greater in isoNAG-dosed perfusions. The comparative results Suggest that isoNAG is a better substrate for adduct formation with liver proteins than NAG.