The effect of age and norepinephrine on renin release by rat kidney slices in vitro.

Basal renin release by rat kidney slices decreases with age in rats of SHR and Sprague-Dawley strains. In contrast, Kyoto-Wistar rats (from which SHR are derived) demonstrate no decrease in renin release with age. The decline in basal renin release observed in SHR occurs at a time when the animal develops hypertension. However, the ability of renin release to respond to stimuli, such as norepinephrine, is enhanced at the time of declining basal renin release and developing hypertension.     

Effects of papaverine on basal and on isoproterenol-stimulated renin secretion from rat kidney slices

Papaverine inhibited the basal renin secretion of rat kidney slices incubated in a physiological salt solution at 37°C. Inhibition was concentration-dependent; secretion was 99 ± 0.2 % inhibited by 5 × 10^?4 M papaverine, and 8 × 10^?5 M was the estimated ED₅₀. In contrast, 2 × 10^?4 M IBMx (3-isobutyl-1-methyl-xanthine) increased the rate of secretion from 215 ± 17 to 366 ± 30 ng hr^?1mg^?1/20 min (p < 0.001). Isoprotenol (4 × 10^?7, 8 × 10^?7, and 5 × 10^?6 M) stimulated renin secretion in a concentration-dependent manner; the stimulatory effects were antagonized by papaverine but unaffected by IBMx. Thus, two known inhibitors of phosphodiesterase--IBMx and papaverine--produce sharply contrasting effects on basal and on isoproterenol-stimulated renin secretion from rat kidney slices.     

The effect of thyroid hormone on renin production and release by rat kidney slices

The effect of thyroid hormone on renin productiona and release by rat kidney slices was studied. Rat kidney slices were incubated in Warburg flasks containing Krebs-Ringer-Phosphate- Glucose- Dextran solution at 37 C for 5 hours. Renin content, renin released into the incubation media and oxygen consumption were measured. Kidney slices actively secreted renin. Kidney slices of hyperthyroid rats released more renin, and kidney slices of hypothyroid rats released less renin than normal kidneys (p less than 0.001). The addition of 1-thyroxine to the incubation medium increased significantly (p less than 0.001) renin release by kidney slices from normal and hypothyroid rats. Thyroid hormone affects renin release through a mechanism independent of the ouabain-sensitive sodium pump and protein synthesis, since ouabain and cycloheximide did not modify renin release or production. The results of this study suggest that thyroid hormone plays a role in renin release from the juxtaglomerular cells.     

Effect of diltiazem, a calcium antagonist, on renin secretion from rat kidney slices

The purpose of these experiments was to characterize the effects of diltiazem on renin secretion from rat renal cortical slices. Incubation of slices in 60 versus 4 mM K medium almost completely abolished renin secretion. Diltiazem antagonized the inhibitory effect in a concentration-dependent manner but had no effect on secretion of slices incubated in 4 mM K medium. Lowering extracellular Ca enhanced the efficacy of diltiazem. These observations demonstrate that Ca influx through voltage-sensitive Ca channels mediates the inhibitory effect of depolarization and further demonstrate that such channels are not open in the basal state of this preparation. In the presence of a concentration of diltiazem which blocked the inhibitory effects of depolarization, both angiotensin II and antidiuretic hormone (ADH) still inhibited secretion. Therefore, both these peptides inhibit secretion by mechanisms which are independent of the voltage-sensitive Ca channels. These observations confirm and extend previous observations suggesting that Ca plays an inhibitory coupling role in the control of renin secretion.     

Hydroxyl radical generation by postischemic rat kidney slices in vitro

To quantitate the formation of hydroxyl radicals (HO.) in ischemia and reoxygenation, dimethyl sulfoxide (DMSO) was added to "trap" evolving HO. in normal, in ischemic, and in ischemic and reoxygenated rat kidney slices, incubated in short-term organ culture in vitro. Hydroxyl radical generation was measured as the accumulation of the specific product of DMSO oxidation by HO., methane sulfinic acid (MSA) in the kidney tissue and surrounding medium using a new colorimetric assay. A mean difference of 7 nmol cumulative HO./gram tissue was detected in rat kidney slices subjected to ischemia and reoxygenation. This amount of HO. generation was not significantly greater than that found in nonischemic or in ischemic but not reoxygenated control tissues, and does not appear to represent the highly toxic burst of HO. radicals implied in current theoretical discussions of reperfusion injury. However, the addition of EDTA chelated iron (1:1) to the incubation medium led to marked postischemic HO. generation. We conclude that clearly toxic numbers of HO. radicals are not formed during reoxygenation in rat kidney slices, either because there is insufficient iron, because only a small fraction of cells in the kidney tissue make oxygen radicals, or because cellular defenses against HO. formation are more powerful than currently appreciated.     

The effects of cyclic AMP, theophylline, angiotensin II and electrolytes upon renin release from rat kidney slices.

The effects of cyclic AMP, theophylline, angiotensin II and electrolytes upon renin release were examined by incubation of rat kidney slices. Angiotensin inhibited renin release with increasing concentrations. On the other hand, cyclic AMP and theophylline stimulated it. Calcium also seemed to play an important role in the control of renin release from kidney slices. However, the direct effects of sodium and potassium upon renin release were not conspicuous.     

A study of the fluid uptake of rat kidney slices in vitro

Rat renal cortical and medullary tissues show a marked elevation of relative water content when immersed in "physiological solutions" containing sodium and chloride ions, or in equally concentrated solutions of monosaccharides. In contrast to this, no increase in relative water content occurs in isosmotic solutions of three disaccharides studied. It appears to be unlikely that the fluid uptake is a result of intracellular hypertonicity existing either physiologically or pathologically. A more satisfactory alternative hypothesis is that ingress of water accompanies entrance of solutes (ions, monosaccharide molecules) into the tissue cells.     

Glomerular podocytes in cultured rat kidney slices

Summary The ultrastructure of rat glomerular epithelial cells (podocytes) in kidney slices in vitro was examined using qualitative and quantitative electron microscopy. The kidney slices were cultured in Medium 199 with Hanks' salts in a 5% CO₂/95% O₂ environment for up to 14 days. Few changes in podocyte ultrastructure occurred in the first 12 h of culture, but by 24 h cell bodies were rounded, microvilli were present on all podocyte surfaces, and some foot processes had been replaced by flattened expanses of cytoplasm. These changes were more pronounced by 3 days, when some podocytes had developed pseudopodal extensions and appeared to be migrating from glomeruli onto the slice surface. Podocytes could still be identified after 8, 10 and 14 days of culture, although relatively few glomeruli remained at 14 days. Morphometric methods were used to analyse podocyte shape, volume and surface area during the first 4 days of culture. The most significant change involved loss of foot processes: the number of filtration slits per 100 m of basement membrane decreased from 211.8 ± 15.0 (mean ± SD) at the commencement of culture, to 55.3 ± 22.6 after 2 days (P < 0.001). These data provide baseline information for in vitro studies on the effects of nephrotoxins on podocytes.     

Different isotonic density gradients in separation of renin granules from rat kidney cortex

Crude renin granule preparations isolated from the rat renal cortex were further purified in isotonic conditions (300 mOsm/kg) using various density gradient materials. It was not possible to separate renin granules from other subcellular organelles using dextran, 40,000-sucrose or metrizamide-sucrose gradients at about 300 mOsm/kg. When osmolality of dextran-sucrose gradients was increased, some separation was found but both renin granules and mitochondria gained density. During a short centrifugation (4640 X g, 30 min) renin granules remained intact and appeared in two populations in Percoll-sucrose gradients. The apparently heavier (larger) particles (at 1.12-1.13 kg/l) were greatly purified from mitochondria (80 X purification vs. the whole homogenate), protein (120 X) and lysosomes (24 X). Electron micrographs demonstrated many dense core granules. The fraction containing apparently lighter (small) granules (at 1.08-1.09 kg/l) was heavily contaminated with mitochondria and lysosomes. During longer centrifugation (4640 X g, 60 min), only one major peak showing renin activity was observed at 1.12-1.13 kg/l, and other cell organelles were lighter. Hence the two renin populations evidently do not differ in density but rather in size. In the animals kept on a low-sodium diet, both types of renin granules were increased.     

Glycine metabolism in rat kidney cortex slices.

We have previously described a method for measuring the rotational diffusion of membrane proteins by using fluorescent triplet probes [Johnson & Garland (1981) FEBS Lett. 135, 252-256]. We now describe the criteria by which the suitability of such probes may be judged. In general, the greatest sensitivity is achievable with probes where the ratio of the quantum yields for prompt fluorescene (phi f) and triplet formation (phi t) are high, as with Rhodamine (phi f/phi t congruent to 10(3)). However, considerations of heat generation at the sample membrane, of time resolution of fast rotations and of irreversible bleaching of the fluorescent probe also apply. The immediate environment of a probe molecule at a membrane protein must also be important in determining the performance of a given probe. Nevertheless, we describe guidelines for evaluating the likely usefulness of fluorescent triplet probes in measurements of membrane protein rotation.     

Purification of rat renal renin from crude kidney extracts by diaminohexamethylene-Sepharose chromatography

Renin from rat kidney extracts was adsorbed to diaminohexamethylene-sepharose columns at extremely low ionic strength and neutral pH. Renin was retarded while the column was developed in 1 mM sodiumpyrophosphate and extraneous proteins were removed. Elution of renin was performed using a linear gradient of sodiumpyrophosphate, 1 – 17 mM at pH 6.8. Renin was purified in a yield up to approx. 60 per cent of the applied activity and a purification factor between 5 – 122 depending on the specific activity of the applied sample. The specific activity after this single chromatography of crude rat kidney homogenate on diaminohexamethylene-sepharose showed a median of 11.3 Goldblatt units per mg protein in a range of 5.3 – 42.0 Goldblatt units per mg protein. The renin binding capacity of the column was 1 Goldblatt unit per ml wet gel. The purified renin was subjected to G-100 Sephadex chromatography demonstrating two molecular weight forms of 44000 and 50000 dalton. Polyacrylamide gel electrophoresis demonstrated three separate fractions of renin.     

The purine nucleotide cycle and ammonia formation from glutamine by rat kidney slices.

To test the significance of the purine nucleotide cycle in renal ammoniagenesis, studies were conducted with rat kidney cortical slices using glutamate or glutamine labelled in the alpha-amino group with 15N. Glucose production by normal kidney slices with 2 mM-glutamine was equal to that with 3 mM-glutamate. With L-[15N]glutamate as sole substrate, one-third of the total ammonia produced by kidney slices was labelled, indicating significant deamination of glutamate or other amino acids from the cellular pool. Ammonia produced from the amino group of L-[alpha-15N]glutamine was 4-fold higher than from glutamate at similar glucose production rates. Glucose and ammonia formation from glutamine by kidney slices obtained from rats with chronic metabolic acidosis was found to be 70% higher than by normal kidney slices. The contribution of the amino group of glutamine to total ammonia production was similar in both types of kidneys. No 15N was found in the amino group of adenine nucleotides after incubation of kidney slices from normal or chronically acidotic rats with labelled glutamine. Addition of Pi, a strong inhibitor of AMP deaminase, had no effect on ammonia formation from glutamine. Likewise, fructose, which may induce a decrease in endogenous Pi, had no effect on ammonia formation. The data obtained suggest that the contribution of the purine nucleotide cycle to ammonia formation from glutamine in rat renal tissue is insignificant.     

In vitro synaptic reconsolidation in amygdala slices prepared from rat brains

The retrieval of consolidated fear memory causes it to be labile or deconsolidated, and the deconsolidated fear memory is reconsolidated over time in a protein synthesis-dependent manner. We have recently developed an ex vivo model where during fear memory deconsolidation and reconsolidation the synaptic state can be monitored at thalamic input synapses onto the lateral amygdala (T-LA synapses), a storage site for auditory fear memory. In this ex vivo model, the deconsolidation and reconsolidation processes of auditory fear memory in the intact brain were prevented following brain slicing; therefore, we could monitor the synaptic state for memory deconsolidation and reconsolidation at the time of brain slicing. However, why the synaptic reconsolidation process stopped after brain slicing in the ex vivo model is not known. One possibility is that brain slicing severs neuromodulatory innervations, which are required for memory reconsolidation, from other brain regions (e.g., noradrenergic innervation). In the present study, we supplemented amygdala slices with exogenous norepinephrine as a substitute for the severed noradrenergic innervations. DHPG (a group I metabotropic glutamate receptor agonist)-induced depotentiation (mGluRI-depotentiation), a marker for consolidated synapses, was observed following norepinephrine application to slices prepared immediately after tone presentation (fear memory retrieval) to rats that had been pre-conditioned to a tone paired with a shock. These results suggest that noradrenergic activation initiates synaptic reconsolidation. In contrast, mGluRI-depotentiation was absent following norepinephrine application to slices that were prepared immediately after the tone presentation (no fear memory retrieval) to rats when a tone and a shock were unpaired, ruling out the possibility that noradrenergic activation somehow facilitates a subsequent synaptic depression induced by DHPG irrespective of synaptic reconsolidation. Furthermore, the restored mGluRI-depotentiation following application of exogenous norepinephrine was dependent on de novo protein synthesis, as is memory reconsolidation. Thus, our findings suggest that T-LA synapses from acute slice preparations can undergo a reconsolidation process, thereby providing an optimal preparation to study a fear memory reconsolidation process in vitro.