Rabbit Kidney Cortex Slices Research Articles

The Efficacy of Antioxidants Administered during Low Temperature Storage of Warm Ischemic Kidney Tissue Slices

Accumulation of products of lipid peroxidation (malondialdehyde, conjugated dienes, lipid peroxides, and Schiff bases) was evaluated in rabbit kidney cortex slices made ischemic for 60 min followed by 18 h storage at 5°C in UW Na gluconate solution and 210 min normothermic reoxygenated incubation. In addition, the effect of adding Trolox (1 mM), deferoxamine (1 mM), and ascorbate (1 mM) as supplemental antioxidants to the UW gluconate solution was evaluated. Lipid peroxidation was slightly increased after hypothermic storage compared to slices subjected to ischemia alone but was not significantly different than ischemic slices during subsequent incubation at normothermia. The addition of either deferoxamine or Trolox to the storage solution substantially reduced lipid peroxidation both during hypothermic storage and subsequent to normothermic incubation. Ascorbate had a mild prooxidant effect as a sole additive to the UW gluconate solution but was clearly prooxidant when combined with either deferoxamine or Trolox. These results suggest that supplemental antioxidants added to the UW gluconate solution under conditions analogous to machine perfusion preservation have a potential role in reducing oxidative stress in kidney tissues harvested after warm ischemia and that hypothermia may be a valuable adjunct to resuscitative therapeutic regimens developed for salvage of ischemic kidneys for transplantation.

Nonesterified Fatty Acid Content of Rabbit Kidney Cortex Slices after Warm Ischemia and Low Temperature Preservation

Nonesterified fatty acid content (NEFA) of rabbit kidney cortex slices preserved at low temperature after warm ischemia was examined. Kidney tissue slices were subjected to 60 min of warm (37°C) ischemia induced by anoxic gaseous phase incubation and then either had reperfusion simulated by normothermic oxygenated aqueous incubation or were preserved at 5°C up to 18 h with UW sodium gluconate solution and then reincubated at normothermia. Slice NEFA content increased during ischemia and remained significantly higher than controls during simulated reperfusion. Ischemic tissue slice NEFA content was significantly reduced by both 3- and 18-h preservations. There was a significant increase in NEFA content in 3-h preserved slices during simulated reperfusion. This increase was relatively unaffected by the addition of KCN to the incubation medium, suggesting that mitochondrial injury was induced by ultrashort preservation of ischemic tissue slices. Ischemic tissue slices preserved for 18 h increased in NEFA content during simulated reperfusion but did not appear more damaged than slices subjected to ischemia alone. Addition of quinacrine (100 μmol/l) to the cold preservation solution significantly reduced NEFA content during simulated reperfusion of slices preserved both 3 and 18 h with a greater effect in 18-h preserved slices. Quinacrine had no effect when added only during simulated reperfusion. KCN addition during simulated reperfusion indicated that quinacrine acted as a mitochondrial protectant in the absence of phospholipase inhibition under these conditions. This study showed that cold preservation may be useful for resuscitation of ischemic tissues harvested for transplantation. Treatments administered during cold preservation of ischemic tissue can have a lasting beneficial effect during reperfusion and may be more effective than similar treatments given only during reperfusion.

Comparative studies of volume restoration following cold-stress induced swelling in renal tissues—I. Effects of ouabain, K+ free medium, colchicine and cytochalasin B on rat and rabbit kidney cortex slices

1. Cold-stress-induced swelling in rabbit and rat kidney cortex slices cannot be due to the sole inhibition of a Na+/K+ exchange system. In these tissues indeed, ouabain induces no swelling and an exchange of Na+ for K+ or a l/l basis. Inhibition of K+ extrusion at low temperature has also to be taken into consideration. 2. Volume restoration at 27 degrees C after cold-stress-induced swelling is inhibited by ouabain in rabbit slices, not in rat ones. The inhibition in rabbit slices is concomitant with an increase in Na+ at levels higher than equilibrium with the external medium. 3. Volume restoration does not seem to implicate colchicine or cytochalasin B sensitive processes.

Comparative studies of volume restoration following cold-stress induced swelling in renal tissues—II. Effects of furosemide, amiloride and K+ permeability in rabbit cortex slices

1. Inhibition of volume restoration by ouabain in rabbit kidney cortex slices is related to an increase in Na+ concentration which may reach levels higher than equilibrium with the external medium. 2. The uphill accumulation of Na+ is not sensitive to amiloride and furosemide. It is dependent on the level of K+ in the external medium. 3. Bringing the slices from cold to normal temperature induces a large increase in Rb86 efflux. 4. It is concluded that the Na+ entry inhibiting volume restoration is driven by an increase in K+ expel occurring upon tissue rewarming.

Secretion control for active and inactive renin: effect of ouabain on release from rabbit kidney cortex slices.

Release of active and inactive renin by rabbit kidney cortex slices was investigated. Inactive renin was estimated as the increase in renin activity after acidification (pH 2.8) of slice supernatant solutions. For kidney slices incubated in complete Krebs bicarbonate buffer, the Na-K-ATPase inhibitor ouabain (100 microM) reduced the secretion of both active (-19.2%) and inactive (-78.9%) forms of renin. In low Na buffers ([Na+] = 23 mM) active renin release was increased and inactive renin was suppressed. Both of these changes were abolished by addition of ouabain (100 microM). The reduction in inactive renin secretion produced by ouabain in complete Krebs buffer did not occur in low [Na+] buffers. In zero Ca2+ buffers containing EGTA (5 mM), secretion of both active and inactive renin was increased but these changes were abolished by addition of ouabain (100 microM). Incubating kidney slices in low Na+, zero Ca2+ media revealed differences between the secretion control mechanisms for the two forms of renin. The separate stimulatory effects of low Na+ and low Ca2+ were not additive for the release of active renin and inclusion of ouabain resulted in similar secretion rates to those under control conditions. For inactive renin secretion, in the absence of Ca2+ release mechanisms still respond to reduction in Na+ with decreased secretion. Conversely, in low Na+ buffers, removal of Ca2+ still promotes inactive renin secretion. These changes were abolished by the addition of ouabain (100 microM). Slices did not change in weight during incubation in media which did not contain ouabain. Addition of this inhibitor to control buffers and low Na buffers did result in an increase in weight. This correlated with the presence of Ca2+ in the buffer and did not appear to be related to [Na+]. These studies again show that the mechanisms regulating the secretion of active and inactive renin are not identical and support the hypothesis that Na+ have differing roles to play in the regulation of these two forms of renin.

Secretion control for active and inactive renin: effects of calcium and potassium on rabbit kidney cortex slices.

Release of active and inactive renin by rabbit kidney cortex slices was investigated. Inactive renin was estimated as the increase in renin activity after acidification (pH 2.8) of slice supernatant solutions. In Ca2+-free media, release of both active and inactive renin was increased but the changes in inactive renin were more marked. The percentage of total renin released which was inactive ranged from 8.3% ([Ca2+] = 9.2 mM) to 34.5% (zero [Ca2+]) with a linear relationship (r = -0.96) over the range of [Ca2+] studied. Depolarizing media ([K+] = 20 mM) suppressed release of inactive renin more than release of the active form. This effect, for both forms of renin, was lost when Ca2+ ions were omitted from the incubation media. This suggests that influx of Ca2+ ions was responsible for the reduced renin secretion following depolarization of the juxtaglomerular cells. Reducing the [K+] of the incubation buffer from 5.7 mM (control) to 1.0 mM did not alter active renin but increased release of inactive renin. Low [K+] media abolished the stimulatory effect of low [Ca2+] on release of both forms of renin. In incubation media with low [Ca2+] or with low [K+] the mixture of renins released by the kidney cortex slices correlated with that found in extracts of non-incubated kidney: that is, about 35% of the total renin was in the inactive form. Mechanisms controlling the secretion of active and inactive renins by the kidney are at least partially independent. Differential secretion of the two forms, perhaps linked to regulation of the activation of inactive renin before release, appears to be the basis of a control step in the over-all expression of the renin-angiotensin system. This may be coupled in some way to juxtaglomerular cell Na+ ion flux.

Effect of ouabain on volume regulation of rabbit kidney cortex slices in hypo-osmotic media

The volume regulation process at work in rabbit kidney cortex slices submitted to hypo-osmotic media show both a swelling limitation and a volume readjustment phase. Swelling limitation is Na+ dependent and is blocked by ouabain 10(-3) M. There is, however, no need to implicate the activity of a ouabain sensitive Na+ /K+ pump in this process.

Effect of osmotic shocks on rabbit kidney cortex slices

Rabbit kidney cortex slices behave an osmometers when withstanding hyperosmotic or hyposmotic shocks of amplitude up to pi 1/pi 2 = 1.25. For hyposmotic shocks of amplitude larger than or equal to pi 1/pi 2 = 1.50, the maximum swelling achieved is less than what can be expected on the basis of the van't Hoff relation, thereby indicating that a volume regulation process is taking place. Volume regulation in kidney slices can be dissociated into two distinct phases. The first one, of swelling limitation, is very rapid and keeps maximum cell volume at values lower than expected when the tissue is considered as an osmometer. This phase is followed by a slow volume readjustment process during which volume progressively decreases towards control values. The major intracellular osmotic effector loss during both swelling limitation and volume readjustment is Na+. The overall volume regulation process is insensitive to furosemide, vanadate, and bumetanide. Swelling limitation is blocked by addition of ouabain. Contrary to what has been believed previously, there is, however, no need to implicate control of the activity of a ouabain-sensitive, Na+/K+ pump in the Na-dependent volume regulation mechanism.

Effects of hypo and hyperosmotic media on rabbit renal cortical slices.

Rabbit kidney cortex slices behave as osmometers when withstanding either hyperosmotic shocks or hypo-osmotic shocks of amplitude up to P1/P2 = 1.25. For hypo-osmotic shocks of amplitude larger or equal to P1/P2 = 1.5 a volume regulation process occurs. Na+ is the main osmotic effector implicated in volume control.

Direct effect of lithium on active and inactive renin secretion.

Rabbit kidney cortex slices were used to investigate the effect of lithium chloride (0 to 20 mm) on renin release. Secretion of active renin was increased in the presence of lithium but inactive (acid‐activatable) renin secretion was unchanged. Increased plasma renin activity in patients receiving lithium therapy may be the result of direct effects of lithium on renin secretion mechanisms rather than being secondary to altered renal sodium handling.

Effects of lipid peroxidation, ca 2+ and adrenalin on the release of fatty acids from rabbit kidney cortex slices

Ascorbic acid (1 mM) plus Fe 2+ (0.4 mM) and Ca 2+ (4 mM) stimulated the release of arachidonic acid from rabbit kidney cortical slices. The release of arachidonic acid was inhibited completely by the addition of mepacrine (1.6 mM). Adrenalin (0.8 mM) enhanced the release of individual free fatty acids (C 16:0, C 18:0, C 18:1, C 18:2 and C 20:4). The release of fatty acids by adrenalin was not inhibited by mepacrine (1.6 mM), but was inhibited by p-chloromercuribenzoic acid (2 mM). These results suggest that the lipolytic process in kidney cortex may be mediated either through the activation of phospholipase A 2 by Ca 2+ and ascorbic acid plus Fe 2+ or through an adrenalin-activated lipase system.

Active and inactive renin release from rabbit kidney cortex slices: effect of sodium concentration and of furosemide.

1. Active and inactive renin release by rabbit kidney cortex slices was investigated. Inactive renin was estimated as the increase in renin activity after acidification (pH 2 . 8) of slice supernatant solutions. 2. Active renin release was increased when incubation medium [Na+] was reduced. This relationship was linear (r2 = 0 . 96) over the range [Na+] = 23-133 mM. 3. For the same range of [Na+] inactive renin secretion decreased when medium [Na+] was reduced (r2 = 0 . 92). Therefore, the proportion of total renin which was in the inactive form decreased linearly as [Na+] was reduced (r2 = 0 . 97). 4. Chloride ions did not appear to be important in altering the secretion of either active or inactive renin. 5. Adding furosemide to the incubation medium in concentrations up to 40 micrograms/ml. did not change secretion of either form of renin. The action of furosemide on secretion of active and inactive renin in vivo is therefore secondary to altered renal function. 6. Regulation of the relative amount of active and inactive renin in plasma could be entirely an intrarenal event. It is not essential to invoke a plasma activating enzyme for inactive renin in order to explain changes in plasma levels of the two forms of renin. 7. This paper supports the hypothesis that release of inactive renin by the kidney is controlled by a sodium-sensitive mechanism.

Secretion control for active and inactive renin: interactions with calcium and sodium ions.

Excitation-secretion coupling for active and inactive renin was investigated by manipulating the ionic composition of media in which rabbit kidney cortex slices were incubated. Alterations in [Ca++] and [K+] produced qualitatively similar, but quantitatively distinct, changes in the release of the two forms of renin. Under control conditions, the slices secreted 15-20% of total renin in the inactive form and exposure to Ca++-free or low [K+] media increased this to 30-40%, a similar proportion to that stored within the kidney. Decreasing [Na+] stimulated release of active renin but reduced inactive renin. There appeared to be antagonism between the effects of Na+ and Ca++ ions in respect of active renin but synergism for release of the inactive form. It is suggested that the characteristics of a single renin release mechanism in the rat kidney resemble those for secretion of inactive, rather than active, renin in other species.

Isoprenaline-induced secretion of active and inactive renin in anaesthetized rabbits and by kidney cortex slices.

1. The effect of the beta-adrenoceptor agonist isoprenaline on the secretion of active and inactive renin was investigated in two preparations. 2. In ten urethane-anaesthetized rabbits isoprenaline, given as a renal artery infusion, had relatively minor effects on renal sodium excretion (increased) and systemic arterial blood pressure (decreased). Urine volume, potassium excretion, creatinine clearance and serum electrolytes were all unchanged. Plasma active and inactive renin both increased immediately and returned to basal values after ceasing the isoprenaline infusion. 3. No significant changes in either plasma renin activity or renal function were observed in a group of ten control animals. 4. The magnitude of the isoprenaline-induced changes in plasma active renin was similar to that in a previous study of frusemide diuresis, but the time course was quite different. Inactive renin disappeared from plasma during frusemide diuresis. 5. Renin release by rabbit kidney cortex slices was also studied. Isoprenaline, added to the incubation medium, caused a dose-related increase in active renin secretion, but inactive renin release remained unchanged. This is in marked contrast to a previous study where reducing [Na+] increased active renin and inhibited inactive renin output. 6. These data support our previous suggestion that activation of inactive renin is regulated by a sodium-sensitive intrarenal mechanism.

Na+-dependent sugar transport in a cultured epithelial cell line from pig kidney.

A Na+-dependent hexose transport system with similar characteristics that observed in the kidney is retained in a cultured epithelial cell line from pig kidney (LLC-PK1). The active transport of oc methyl-D-glucoside (oc MGP), a nonmetabolizable sugar, which shares the glucose-galactose transport system in kidney cells is mediated through a Na+-dependent, substrate-saturable process. The kinetic analysis of the effect of Na+ on the uptake of ocMGP indicated that the Na+-sugar cotransport system is an affinity type system in which the binding of either sugar or Na+ carrier increases the affinity for the other ligand without affecting the Vmax. The sequence of selectivity for different sugars studied by the inhibition produced in the uptake of ocMGP is very similar to that reported in rat kidney, rabbit kidney cortex slices, and rabbit renal brush border membrane vesicles. Phlorizin, even at very low concentration, almost completely inhibits ocMGP uptake. Conversely, phloretin at the same low concentration stimulated the sugar accumulation by inhibition of efflux, probably at the level of the basolateral membrane. Sulfhydryl group inhibitors also blocked the ocMGP uptake, suggesting that these groups were required for normal functioning of the sugar carrier system. This sugar transport system is an important functional marker to study the molecular events associated with the development of polarization in epithelial cells.

Sugar, Amino Acid, and NA+ Cotransport in the Proximal Tubule

For obvious reasons the Na+-dependence of sugar and amino acid transport was described much earlier for the intestine (5, 15, 19, 61, 62) than for the kidney. Thus, the Na+-sugar cotransport hypothesis was already well for­ mulated (15) before the sodium dependence of renal glucose transport had been reported in perfused rat (64) and frog kidney (91, 92) and in rabbit kidney cortex slices (52). Similarly, the sodium dependence of renal amino acid transport was studied by uptake into tubular cells (24, 40, 41, 69, 70, 71, 79) before the introduction of new techniques, such as the doubly perfused proximal tubule in situ (86), with transtubular or cellular electrical potential measurements (30) and the preparation of osmotically reactive plasma membrane vesicles from either cell side (51), made it possible to study the single transport steps and to test the sodium gradient hypothesis rigorously. The vesicular transport studies have already been reviewed extensively (48, 65).

The effects of cyclic nucleotides and some related agents on 32Pi labeling of renal polyphosphoinositides in Vitro

When rabbit kidney cortex slices were incubated in the presence of 32 P i and dibutyrylcyclic AMP (dbcAMP) 4 a significant decrease in the labeling of phosphatidyl inositol phosphate (DPI) but not phosphatidyl inositol bisphosphate (TPI) was observed. In the presence of 0.3 m m caffeine cyclic AMP (cAMP) produced a similar effect. Caffeine potentiated the inhibitory effect of dbcAMP. At high concentrations (3 m m) caffeine alone decreased the 32 P i labeling of both DPI and TPI. These decreases in 32 P i labeling were not mediated by decreases in the labeling of intracellular P i or ATP as measured by 10-min acid-labile nucleotide phosphate (10′-ALNP). Addition of cyclic GMP (cGMP) to the incubation medium decreased the labeling of DPI and to a lesser extent that of TPI also. Addition of parathyroid hormone (PTH) to the incubation medium (in the absence of exogenous cyclic nucleotides) also decreased the 32 P i labeling of DPI but not that of TPI. In contrast to the effects of cAMP, dbcAMP, cGMP, PTH, and caffeine, the addition of insulin to the incubation medium resulted in increased 32 P i labeling of DPI with no effect on TPI labeling. DPI isolated from kidney cortex slices prelabeled with 32 P i and subsequently incubated with cAMP or dbcAMP contained less label than DPI isolated from slices similarly prelabeled but subsequently incubated in the absence of either cAMP or dbcAMP. These data suggest an increased rate of DPI breakdown in the presence of elevated cAMP or dbcAMP concentrations. This hypothesis was supported by the fact that cAMP stimulated the hydrolysis of DPI but not of TPI by a polyphosphoinositide phosphodiesterase present in the supernatant fraction of rabbit kidney cortex.

Concentrative PAH transport by rabbit kidney slices in the absence of metabolic energy.

Characteristics of para-aminohippurate (PAH) transport in the absence of intracellular metabolism were studied with Na+, K+-depleted and ouabain-poisoned rabbit kidney cortex slices. The imposition of a NaCl gradient (out to in) resulted in specific stimulation of PAH uptake. PAH accumulated against its concentration gradient when cell [Na+] was less than medium [Na+]. Conversely, renal cells extruded PAH when cell [Na+] exceeded medium [Na+]. Membrane potential measured with triphenylmethylphosphonium revealed that conditions which created an interior-positive membrane potential inhibited the Na+-dependent transport of PAH but caused stimulation of the Na+-independent component. Characteristics of the Na+-dependent PAH transport system in ouabain-poisoned slices were similar to those previously described in metabolically active tissues.

Maturation of p-aminohippuric acid transport in the developing rabbit kidney: interrelationships of the individual components.

The excretion of both endogenous organic (aryl) acids, such as benzoic acid, and exogenous ones, such as p-aminohippuric acid (PAH), penicillin, and furosemide, is reduced in the human neonate and other immature animals. The unique developmental pattern of aryl acid transport in immature rabbit kidney cortex slices is produced by the interrelationships of PAH uptake, efflux, and amount of intracellular binding protein, all of which reach mature levels at different ages.

Accumulation of <i>p</i>-Aminohippurate by Multiple Transport Systems in Rabbit Kidney Cortex Slices: A Kinetic Analysis

A kinetic analysis of the rate of active (oxygen-dependent) accumulation of <i>p</i>-aminohippurate (PAH) by rabbit renal cortical slices was performed to confirm the existence of multiple renal organic anion transport systems. The kinetic analysis demonstrated the presence of two active uptake modes for PAH in this slice system. Acetate increased the rate of the oxygen-requiring component of each mode of PAH uptake by increasing the apparent V<sub>max</sub> of transport. Additional PAH transport modes were not revealed by the addition of acetate. These results were consistent with the hypothesis that more than one active transport system participates in the transport of organic anions in the renal proximal tubule cell.