Dog Kidney Slices Research Articles

Species differences of organic anion transporters involved in the renal uptake of 4‐amino‐3‐chlorophenyl hydrogen sulfate, a metabolite of resatorvid, between rats and dogs

Previous studies on the metabolic fate of resatorvid (TAK-242) have shown that species differences in the pharmacokinetics of 4-amino-3-chlorophenyl hydrogen sulfate (M-III), a metabolite of TAK-242, between rats and dogs are mainly attributable to the urinary excretion process. In the present study, the renal uptake mechanism of M-III was investigated using kidney slices and Xenopus laevis oocytes expressing rat organic anion transporter 1 (rOat1; Slc22a6) and rOat3 (Slc22a8). The uptake of p-aminohippuric acid (PAH), a substrate for Oats, by kidney slices from rats and dogs increased at 37 °C and M-III inhibited the uptake. The initial uptake clearance of M-III by rat kidney slices was 0.295 and 0.0114 ml/min/g at 37 °C and 4 °C, respectively. The Eadie-Hofstee plot of M-III uptake at 37 °C revealed two-component transport processes with K(m) values being 6.48 and 724 µmol/l. The uptake was inhibited by probenecid (PBC), PAH and benzylpenicillin (PCG). In contrast, in dog kidney slices, the initial uptake clearance of M-III was 8.70 × 10(-3) and 9.00 × 10(-3) ml/min/g at 37 °C and 4 °C, respectively, and the uptake was not inhibited by PBC. Furthermore, rOat1- and rOat3-expressing oocytes mediated M-III uptake and the uptake was inhibited by PAH and PCG, respectively. These results suggest that rOat1 and rOat3 are responsible for the renal uptake of M-III in rats. Moreover, it is speculated that Oat(s) is unable to transport M-III in dogs and that the difference in the substrate recognition of Oat(s) contributes to the species difference in the pharmacokinetics of M-III between rats and dogs.

Biotransformation of the antiemetic 5-HT3 antagonist tropisetron in liver and kidney slices of human, rat and dog with a comparison to in vivo.

Species differences in the biotransformation of the antiemetic tropisetron, a potent 5-hydroxytryptamine type 3 (5-HT3) receptor antagonist, were evident in liver slice incubates of human, rat and dog, and reflected the species differences observed in vivo with respect to the relative importance of individual pathways. The dominant biotransformation pathway of tropisetron (10 microM) in human liver slices was formation of 6-hydroxy-tropisetron, whereas in rat liver slices it was 5-hydroxy-tropisetron, and in dog liver slices N-oxide formation. Initial rates of tropisetron metabolite formation in the liver slices (8 mm in diameter, 200 +/- 25 microns thickness) of human (83 +/- 61 pmol/h/mg slice protein), rat (413 +/- 98 pmol/h/mg slice protein) and dog (426 +/- 38 pmol/h/mg slice protein) would predict less of a first-pass effect in humans compared to the rat or the dog. For human and rat, the prediction matched well with the species ranking of tropisetron bioavailability; however, for dog the in vitro data overestimated the apparent first-pass effect. The jejunum is not expected to contribute to the first-pass effect in humans, since human jejunum microsomes did not metabolize tropisetron. The major organ of excretion for tropisetron and its metabolites is the kidney, but the contribution of the kidney to the overall metabolism of tropisetron would be small. Species independent N-oxide formation (2-12 pmol/h/mg slice protein) was the major pathway in human, rat and dog kidney slices, and was comparable to N-oxide formation in the rat and human liver slices but was 1/10 the rate in dog liver slices. This study has demonstrated that the liver is the primary site of tropisetron biotransformation, and the usefulness of organ slices to characterize cross species differences in the dominant biotransformation pathways.

Cold- and Cryopreservation of Dog Liver and Kidney Slices

The use of tissue slices in culture could decrease the number of animals used in health-related research and decrease experimental variation. This reduction may come about particularly if the methods of cold- and cryopreserving tissue slices are perfected, and one can conduct sequentialin vitroexperiments into xenobiotic metabolism, organ-specific toxicity, or organ-specific biochemical processes with tissue slices. With this goal in mind, dog liver and kidney slices were placed in cold storage at 0°C using Viaspan (UW), Euro–Collins (EC), Sacks + prostacyclin (SP), and V-7 (V7) cold-preservation solutions for 10 days. Viability was assessed each day by measuring K+content and protein synthesis after 4 h of incubation in Waymouth + 10% fetal calf serum (FCS). Dog liver slices can be cold-preserved in V7 for up to 7 days using K+retention as the viability criterion but only up to 4 days using protein synthesis. Dog kidney slices can be cold-preserved in UW, EC, and V7 for up to 10 days using K+retention, but only V7 could maintain protein synthesis for 10 days. Cryopreserved dog liver and kidney slices retained 63–68% of control viability after 4 h of incubation in FCS. The cryopreservation regimen included using 10% dimethyl sulfoxide in FCS as the cryoprotectant, a freezing rate of 0.5°C/min for liver slices and 12°C/min for kidney slices, and thawing in 37°C FCS. Continued development of cold- and cryopreserving tissue slices could reduce the numbers of animals used and provide accurate and reproducible data.

Transmembrane Fluxes of Potassium in Dog Kidney Slices

The effect of warm ischemia on the transmembrane transport of potassium in dog kidney slices was studied by measurement of the uptake of 42K. The requirement for steady-state conditions concerning the intracellular potassium concentration was thereby studied. The total potassium content in the slices was found to be constant between 120 and 180 min incubation at both 25 and 37 degrees C. The cell water calculated from the total tissue water and 14C-inulin space in the dog kidney slices amounted to 38 ml-100 g wet weight-1 at 37 degrees C and 45 ml-100 g wet weight-1 at 25 degrees C and was found to remain constant for the incubation interval 120--180 min. The major part of the tissue uptake of 42K could be described by one single mono-exponential function under these conditions. The transmembrane influx at 37 degrees C calculated by using a modified Keynes formula amounted to 1.70 mmol K+-kg wet weight-1-min-1 after no warm ischemia and to 0.89 mmol K+-kg wet weight-1-min-1 after 2 h warm ischemia. The corresponding values for incubation at 25 degrees C were 1.26 and 0.77 mmol K+-kg wet weight-1-min-1, respectively. In the slices incubated at 25 degrees C, the potassium content was higher and the sodium content lower than in slices incubated at 37 degrees C.

Inhibition by Iodide of the Activation of the Thyroid Cyclic 3′,5′-AMP System

The action of iodide on the cyclic AMP system of dog thyroid slices has been studied. Iodide inhibits the enhancement of cyclic AMP accumulation in the presence of TSH. Such an effect is also observed in horse, beef and sheep thyroid slices, but not in dog kidney slices stimulated by parathyroid hormone or in rat parotid slices stimulated by isoproterenol. The effect in dog thyroid slices is suppressed by 1mM NaClO4, 1mM methimazole and 1mM propylthiouracil. Similar data have been obtained for prostaglandin E1 stimulation. Effects of thyrotropin mediated by cyclic AMP, i.e., activation of iodothyronine secretion, 1-14C-glucose oxidation, and lactate formation, were also inhibited by iodide but not by iodide and methimazole. Similar activations when caused by dbcAMP were not inhibited by iodide. The data suggest a model in which an intracellular agent resulting from the oxidation of iodide acts on the thyroid cyclic AMP system.

Effects of Warm Ischemia on the Transmembrane Fluxes of Potassium in Dog Kidney Slices

The influence of warm ischemia on the transmembrane transport of potassium ions in dog kidney slices was studied by measurements of the uptake and washout of 42K. The tracer ion equilibrate

Effects of warm ischemia on the potassium and sodium contents and on the sodium-, potassium-ATPase activity in dog kidney slices.

The potassium and sodium contents in dog kidneys were studied after various warm ischemia times. A considerable net loss of potassium as well as a gain of sodium were demonstrated during the first hou

Kinetics of inulin penetration in cortex and medullary kidney slices.

The inulin space of dog kidney slices obtained from cortex and outer and inner medulla was evaluated by incubating the tissue at different time intervals in inulin Ringer. When the kidney was cut normal to the major axis of Henle's loops and collecting ducts, inulin uptake appeared to follow a single exponential time function in each renal region.When the organ was cut parallel to the major axis of Henle's loops the inulin space was greater and inulin uptake appeared to follow two exponential functions of time in each of the three renal regions. This would indicate that the plane of section can influence the apparent size of the extracellular space of kidney slices, as measured by the distribution of inulin.

Metabolic and functional effects of acute renal ischemia in dog kidney slices.

Metabolic and functional effects of acute renal ischemia in dog kidney slices.

The inhibition of glycine and β-methylglucoside transport in dog kidney cortex slices by ouabain and ethacrynic acid: Contribution to the understanding of sodium-pumping mechanisms

1. 1. In an attempt to provide confirmatory evidence for the existence of dual sodium pumps in dog kidney slices, the inhibition of glycine and β-methylglucoside uptake by ouabain and ethacrynic acid, the classic inhibitors of the two pumps, was studied. 2. 2. The active accumulation of these two substrates was entirely Na +-dependent, and was partially inhibited by high concentrations of each of the inhibitors. A mixture of the inhibitors at high concentrations completely suppressed active transport of the substrates. Studies on radiosodium uptake confirmed that the locus of action of the drugs is centred on the sodium-pumping mechanisms. 3. 3. In conjunction with studies on Na +K +-ATPase, which in this tissue is insensitive to ethacrynic acid, these findings are consistent with the two-pump hypothesis for sodium transport in the proximal tubule.

A RELEASE OF RENIN FROM DOG KIDNEY CORTEX SLICES

A release of renin from kidney cortex slices in vitro was first reported by the present authors (1). Recently, the renin release from kidney slices of rats, pretreated with deoxycorticosterone acetate, clipping of renal artery, bleeding or decapitation, was shown by Jong (2) and BOZOVIC et al. (3). In their experiments, the fundamental process of renin release has not been made clear. In our previous paper, it was pointed out that renin is released from dog kidney slices into an incubation medium depending on the temperature and pH of the medium and about 10% of renin contained in the original slices is released within 60 minutes of incubation. Presently, the effects of metal ions in the incubation medium and atmosphere on renin release were studied. From the present findings, it is assumed that renin is released, partialy included in the renin granule, into the incubation medium. Further study was made on the release of several enzymes found in other cell particles to compare the releasing pattern of renin from slices.

Alpha-ketoglutarate metabolism and K+ uptake by dog kidney slices.

Alpha-ketoglutarate metabolism and K+ uptake by dog kidney slices.

INFLUENCE OF ANALOGUES OF PHENYLBUTAZONE ON RENAL TRANSPORT OF 4-AMINOHIPPURATE.

Analogues of phenylbutazone were tested for inhibitory potency in the renal transport mechanism for 4-aminohippurate. In a series of ten compounds examined by an in vitro technique, no correlation could be demonstrated between inhibitory potency and acidic strength of the inhibitor either in rabbit or in dog tissues. In a further series of 16 paired analogues, the hydroxylated member of each pair was 3–4 times less potent than its unhydroxylated counterpart. Correlations between molecular structure and pharmacological activity are suggested. In contrast to these observations, each of three selected phenylbutazone analogues (phenylbutazone, metabolite II, and sulfinpyrazone) depressed the maximum tubular excretory rate for 4-aminohippurate in intact dogs, but apparent inhibitory potencies by the clearance technique were unrelated to inhibitory potencies in dog kidney slices. Differences in relative potencies thus obtained are attributed to differences in metabolic alteration of each compound at extrarenal sites in intact dogs.