Rat Renal Cortical Cells Research Articles

Cytokines and the hypothalamic-pituitary-adrenal axis

After administration of the cytokines interleukin 1 (IL1), tumor necrosis factor (TNF), interleukin 2 and interleukin 6 to laboratory animals or humans, plasma levels of glucocorticoids are elevated. This effect is mediated by activation of the hypothalamic-pituitary unit. IL1 and TNF inhibit aldosterone production by rat adrenocortical cells in vitro and stimulate renin release by rat renal cortical cells. Administration of IL1 or TNF in rats suppresses hypothalamic-pituitary-thyroid function, whereas IL1 acts at the level of the brain and the gonads to interfere with gonadotropin and sex steroid secretion. During stimulation of the immune system (e.g. during infectious diseases), peculiar alterations in hormone secretion occur (hypercortisolism, hyperreninemic hypoaldosteronism, euthyroid sick syndrome, hypogonadism). The role of cytokines in these alterations remains to be established.

Inhibitory effect of endothelin on renin release in vitro.

We previously reported that endothelin inhibits renin release in a dynamic superfusion system of collagenase-dispersed rat renal cortical cells. In the present report we investigated cellular mechanisms by which endothelin inhibits renin release from juxtaglomerular (JG) cells. In a superfusion system of dispersed rat renal cortical cells, 10(-10) M endothelin inhibited renin release stimulated by 5 x 10(-8) M isoproterenol or 5 x 10(-5) M 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8, a putative intracellular Ca antagonist). Endothelin showed a slight but significant inhibitory effect on renin release stimulated by isoproterenol or TMB-8 even in the absence of extracellular Ca. Endothelin also inhibited renin release in the presence of 10(-4) M nicardipine. In a superfusion system of renal cortical slices, both 10(-8) M endothelin and a high concentration (60 mM) of K inhibited renin release, and 10(-6) M nicardipine attenuated the inhibition of renin release by high K but did not affect the inhibition by endothelin. These results suggest that endothelin inhibits renin release from JG cells not only by the promotion of Ca influx into the cells through dihydropyridine-insensitive Ca channels but also by other mechanism(s) independent of extracellular Ca.

Inhibitory Role of Ca2+ in the Control of Renin Secretion: A Study Using Supervised Dispersed Rat Renal Cortical Cells

1. The role of Ca2+ in the control of renin release was investigated using a collagenase-dispersed rat kidney cortex cell preparation. 2. Superfusion with a series of low [Ca2+] buffers in either ascending or descending order of concentration increased renin release. Exposure to 0.06 mmol/l Ca2+ increased release by 120% (P less than 0.001) when presented as the first buffer in ascending order of concentration and by 79% (P less than 0.001) when presented as the fourth and last in a series of descending order. 3. The Ca2+ entry blocking drug diltiazem in a range of concentrations increased renin release and at 10(-5) mol/l diltiazem the mean stimulation was 35% (P less than 0.01). 4. 8-(N.N-Diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) reduces the release of Ca2+ from intracellular stores and, studied over a range of concentrations, this compound increased renin release. At 10(-5) mol/l TMB-8 the mean increase was 44% (P less than 0.001). 5. None of these experimental manipulations, low [Ca2+], diltiazem or TMB-8, had any effect on the release of adenosine 3':5'-cyclic monophosphate into the cell superfusate, indicating that a decrease in intracellular [Ca2+] increases renin release by a mechanism which is independent of changes in adenosine 3':5'-cyclic monophosphate production. 6. Effects of low [Ca2+], diltiazem and TMB-8 on renin secretion were all shown to be reversible when superfusion with control buffer was resumed.

Application of a novel radioimmunoassay for serotonin in the detection of aromatic l-amino acid decarboxylase activity in isolated renal cortical cells

The decarboxylation of amino acids is an cssential step in the biosynthesis of biogenic amines; the enzyme aromatic Lamino acid decarboxylase (AAAD; EC 4.1.1.28) catalyses the decarboxylation of both ~-3,4-dihydroxyphenylalanine and L-5-hydroxytryptophan (5-HTP) to form dopamine or serotonin (5-HT), respectively. The AAAD enzyme is present in many tissues (Rahman et ul., 1 9 8 1 ), but is particularly active in the kidney, where the products of decarboxylation (i.e. dopamine and 5-HT) are thought to affect renal haemodynamics directly. Dopamine causes marked natriuresis, diuresis, and dilatation of the renal vasculature, whereas 5-HT is thought to exert opposite effects (Stier et al., 1984). Several previous studies have measured AAAD activity by incubating tissue homogenates with one of the enzyme substrates, followed either by direct measurement of the mass of product generated in the incubate, using high-pressure liquid chromatography coupled with electrochemical detection (Stier et al., 1987) or by measuring the amount of C 0 2 produced during the decarboxylation reaction (Clark et d., 1954). Both techniques are time consuming, and therefore restrict the throughput of large numbers of samples. We have used a radioimmunoassay ( M A ) for 5-HT developed in our own laboratories (Gow et al., 1987) to measure the production in vitro of 5-HT from 5-HTP by isolated rat renal cortical cells (RCC). Female Wistar rats (200-250 g) were killed by cervical dislocation and the kidneys removed immediately into icecold physiological saline. The kidneys were trimmed of fat, the capsules removed, and the cortex dissected from the medulla. Intact RCC were prepared using the collagenase digestion procedure described by Drury et ul. (1986), with modifications by Noble et al. (1988). The cells were finally resuspended in Medium 199 containing bovine serum albumin ( 2 g/l). For each experiment, aliquots (800 pl ) of a freshly prepared RCC suspension containing 0.5 x 1 O6 cells/ml were dispensed in duplicate into glass tubes in an ice/water slurry (4C), followed by Medium 199 (100 pl) containing both pargyline (final concentration 1 W 6 mol/l), and varying doses of 5-HTP ( O l O s mol/l, final concentration). A further aliquot of Medium 199 (100 p l ) or, for the inhibitor experiments, Medium 199 containing the AAAD inhibitor carbidopa (100 pl; lo-' mol/l, final concentration) was added, and the tubes incubated in a shaking water-bath (120 cycles/min) at 37°C for 30 min. The reaction was stopped by plunging the incubation tubes into an ice/water slurry, followed by centrifugation at 1720 g for 20 min at 4°C. The supernatants were then collected, and frozen at 20°C until analysis by RIA. Our results (Fig. 1) show a dose-dependent production of 5-HT by RCC in response to the increasing concentration of substrate (5-HTP). This response was significantly diminished ( P < 0.05, 2-way analysis of variance) by the inclusion of carbidopa ( lo- mol/l) in the incubates and is in agreement with the observations of Stier & ltskovitz (1985).

Locally formed dopamine inhibits Na+-K+-ATPase activity in rat renal cortical tubule cells.

Dopamine, generated locally from L-dopa, inhibits Na+-K+-ATPase in permeabilized rat proximal tubules under maximum transport rate conditions for sodium. To determine whether locally formed dopamine inhibits Na+-K+-ATPase activity in intact cortical tubule cells we studied the effect of L-dopa on ouabain-sensitive oxygen consumption rate (QO2) and 86Rb uptake in renal cortical tubule cell suspensions. L-Dopa (10(-4) M) did not affect ouabain-insensitive QO2 or mitochondrial respiration. However, L-dopa inhibited ouabain-sensitive QO2 in a concentration-dependent manner, with half-maximal inhibition (K0.5) of 5 x 10(-7) M and a maximal inhibition of 14.1 +/- 1.5% at 10(-4) M (P less than 0.05). L-Dopa also blunted the nystatin-stimulated QO2 in a concentration-dependent manner, with a K0.5 of 5 x 10(-8) M and a maximal inhibition of 21.8 +/- 1.2% at 10(-5) M (P less than 0.05), indicating that L-dopa directly inhibits Na+-K+-ATPase activity and not sodium entry. Ouabain-sensitive 86Rb uptake was also inhibited by L-dopa (16.0 +/- 2.4%, P less than 0.05). Carbidopa (10(-4) M), an inhibitor of the conversion of L-dopa to dopamine, eliminated the effect of L-dopa on ouabain-sensitive QO2 and 86Rb uptake, indicating that dopamine rather than L-dopa was the active agent. The finding that the L-dopa concentration-response curve was shifted to the left by one order of magnitude in the presence of nystatin suggests that the inhibitory effect is enhanced when the intracellular sodium concentration is increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies of structure-function relationship of human parathyroid hormone using rat and human renal cortical cells in culture.

The structure-function relationship of human PTH was studied by testing the effects of its synthetic analogues on cAMP production in cultures of rat and human renal cortical cells. The human cell system was by two orders more sensitive to an agonist, hPTH1-34, than the rat cell system. In both culture systems, Tyr34hPTH3-34NH2, which by itself had no influence on adenylate cyclase, caused approximately 90% inhibition of the hPTH 1-34-induced cAMP production at a molar ratio of 1000:1. On the other hand, Tyr34hPTH5-34NH2, Tyr34hPTH7-34NH2, Tyr34hPTH8-34NH2 and Tyr34hPTH9-34NH2 had weak but distinct agonist activity. When these four analogues were tested in the presence of hPTH1-34, they acted as antagonists in the human cell system, whereas they still showed agonist activity in the rat cell system which was additive to that of hPTH1-34. These data indicate that the amino acids at positions 1 and 2 of hPTH are important for its biological activity and that those in the sequence 9-34 also participate in the activation of adenylate cyclase. The higher sensitivity to hPTH1-34 and the different response to some of the analogues of human renal cortical cells compared with rat renal cortical cells emphasize the importance of employing human cell systems in evaluating the structure-function relationship of hPTH.

Renotropic activity of lutropin: Direct stimulation of DNA synthesis of cultured rat renal cortical cells

Two differently purified ovine lutropin (LH) preparations were studied to see if they stimulated [ 3H] thymidine incorporation into cultured rat renal cortical cells. Both preparations showed a dose-dependent (0.11–1 ng/ml), time-dependent (peaking at 18 h), serum-dependent (7.5% of fetal calf serum or 10% castrated-hypo-physectomized rat serum) renotropic effect. Ovine TSH and FSH failed to mimic this specific, renotropic effect. We concluded that LH directly stimulates renal DNA synthesis in cooperation with other serum factor's.

Development and application of a superfusion technique for the study of renin secretion in rat renal cortical cells.

A dynamic column superfusion system has been developed for the study of renin secretion in rat renal cortical cells. Cells were isolated by collagenase digestion and mechanical dispersion, before suspension with polyacrylamide beads and superfusion with oxygenated physiological medium. Renin was detected in the superfusate by incubation of fractions with excess nephrectomized sheep substrate in the presence of angiotensinase inhibitors followed by radioimmunoassay of the angiotensin I generated. Optimized methodology included a purpose-built polytetrafluorethylene flow cell, a 1 h equilibration to achieve a steady state, 5 min eluate collections, a 15 min stimulatory and a 30 min recovery period, and duration of perfusion of up to 270 min. Significant increments above baseline renin release were seen with the stimuli of adrenaline, noradrenaline and isoprenaline. These could be demonstrated with concentrations of 10(-9) mol/l (adrenaline), 5 X 10(-10) mol/l (noradrenaline) and 10(-9) mol/l (isoprenaline). This technique has significant advantages over previous methods for the study of renin secretion in vitro at the cellular level. It is reproducible and sensitive, and avoids many of the limitations of static cell suspension and kidney slice methods.

Factors regulating prostaglandin E 2 biosynthesis in renal cortical tubular cells

Prostaglandin synthesis by isolated rat renal cortical tubular cells was studied in vitro with a superfusion system. The cells were introduced in Teflon chambers and intermittently stimulated. The PGE 2 production was measured in the effluent. ANG II (10 −10–10 −6M) induced a dose-dependent increase in PGE 2 synthesis. Saralasin antagonized the response to ANG II. Hyperosmolar mannitol or NaCl and Ca 2+-ionophore A23187 also stimulated PGE 2 synthesis. The PGE 2 response to all stimuli was blocked in Ca 2+-free media containing EGTA. The Ca 2+-channel blocker nifedipine (10 −10–10 −6M) did not significantly inhibit the PGE 2 response to ANG II, hyperosmolar mannitol or NaCl, and A23187, whereas the phospholipase-inhibitors p-bromophenacyl bromide (10 −4M) and chloroquine (10 −4M) inhibited the response. Thus, PGE 2 synthesis in response to these stimuli in rat renal cortical tubular cell is a Ca 2+-dependent process, acting via phospholipases by a mechanism which does not appear to involve voltage-dependent Ca 2+ -channels.

Effect of Calcium, Sodium and Isoproterenol on Renin Secretion From Disaggregated Rat Renal Cortical Cells

A disaggregated cell system prepared from rat renal cortices has been developed to study the regulation of renin release in vitro. Cell suspensions were prepared by incubating minced renal cortical tissue in collagenase. The digested tissue was filtered sequentially through graded Teflon meshes of 125 and 44 mu, respectively. This preparation contained less than 2% intact glomeruli or tubules. Incubation of cell suspensions with the beta-adrenergic agonist L-isoproterenol (ISO) significantly increased the activity of renin released into the incubation media. Peak levels of renin activity were detected 10 to 15 min after the addition of ISO. The apparent ED50 for stimulation of renin release by ISO was 6 X 10(-8)M and the response was antagonized by the beta-adrenergic antagonist dl-propranolol. The spontaneous release of renin was also suppressed by increasing the concentration of extracellular calcium, whereas sodium ions had no effect on this process. These data have demonstrated that disaggregated renal cortical cell models are useful for studying the in vitro effects of pharmacologic agents and ions on renin secretion.

Stimulation of thymidine and uridine incorporation into rat renal cortical cells in primary monolayer culture by uninephrectomized rat serum.

The effects of sera from uninephrectomized rats or sham-operated rats on the facilitation of 3H-thymidine incorporation into the cells were examined in the primary monolayer culture of rat renal cortical cells. At serum concentrations ranging from 0.25 to 5.0%, a significant and identical stimulation of the thymidine uptake was seen with both sera. The difference in facilitating the thymidine uptake between the sera from the uninephrectomized and sham-operated rats was significant at a serum concentration of 10%. The compensatory renal growth after uninephrectomy may not be due to the simple elevation of the growth factors that are present in the normal serum, but may be due to the renotropin induced by the reduced renal mass.

Amiloride inhibits mammalian renal kallikrein and a kallikrein-like enzyme from toad bladder and skin.

Renal kallikrein is localized in luminal plasma membranes of the mammalian distal nephron and gains access to urine from this site. Its activity is regulated, in part, by aldosterone. These facts led us to study the effects of amiloride, a drug known to inhibit sodium reabsorption and potassium secretion at this site, on kallikrein activity. Amiloride inhibited the esterolytic activity of purified rat or human urinary kallikrein or of rat renal cortical cells upon a synthetic substrate (ID50 = 0.12-0.23 mM). Kinetic analyses showed that the enzyme inhibition was noncompetitive and reversible in nature. The kinin-generating activity of kallikrein acting upon kininogen substrates was also inhibited by amiloride, as measured by bioassay in the rat uterus of guinea pig ileum or by radioimmunoassay of liberated kinins (ID50 = 85 microM). No other diuretic drug tested inhibited kallikrein activity, except triamterene, which did so, weakly. In addition, kallikrein-like enzyme activity was discovered in the urinary bladder or skin of Bufo marinus toads and this activity was also inhibited by amiloride. The localization of the enzyme and its inhibition by this drug suggest that further study of relationships amongst the glandular kallikrein-kinen system and renal ion and water transport is warranted.

The effects of aldosterone and spironolactone on renal kallikrein in the rat.

1. A technique has been developed for the measurement of kallikrein 'production' in rat renal cortical cells in suspension. 2. After preparative steps, column chromatography on DEAE-cellulose yielded a peak of alpha-N-tosyl-L-arginine methyl ester (Tos-Arg-OMe) esterase activity identical with kallikrein isolated from rat urine in respect of pH optimum, effects of inhibitors, biological activity and immunological properties. 3. The nutrient medium surrounding incubated cells contained measurable kallikrein activity, which was increased by aldosterone and decreased by spironolactone. 4. The results raised the possibility that kallikrein could be an aldosterone-induced protein.

Studies on rat renal cortical cell kallikrein I. Separation and measurement

A technique has been developed to separate and measure kallikrein in a heterogeneous population of rat renal cortical cells in suspension. After rat kidneys were perfused in situ in anaesthetized rats, viable, counted cortical cell suspensions were obtained. Cells were suspended in a sucrose/Tris buffer containing 0.5% deoxycholate, homogenized, centrifuged, dialyzed, and gel filtered on Sephadex G-25. Column chromatography on DEAE-cellulose resulted in a single peak of esterase activity between 0.20 to 0.25 M NaCl/sodium phosphate buffer. Subsequent elution yielded an alkaline esterase which was identical to kallikrein isolated from rat urine, insofar as pH optimum, effects of inhibitors, bioassay activity and immunological properties were concerned. Calculated yields were about 70% of the total esterase activity present in the parent cell homogenates. Recoveries of a purified rat urinary kallikrein added to the cell homogenates, the DEAE-cellulose columns, or the eluates from the columns ranged from 83–108% (mean 96%). Using this technique, it was found that the amount of kallikrein activity present in non-incubated renal cortical cells ranged from 0.6 · 10 −2 to 4.6 · 10 −2 α-N- tosyl- l-arginine methyl ester (Tos-Arg-OMe) esterase units per 10 8 cells. However, cells incubated in a nutrient medium at 37°C for 3–8 h contained no measurable kallikrein activity, whereas the surounding medium had kallikrein activity which could be significanyly decreased by aldosterone and decreased by spironolactone.