Renal Microsomes Research Articles

Solubilization of a phospholipid-requiring enzyme, iodothyronine 5′-deiodinase, from rat kidney membranes

Enzymic activities catalyzing the reductive 5′-deiodination of thyroxine and 3,3′,5′-triiodothyronine were solubilized from rat kidney microsomes by treatment with 0.2% deoxycholate. Deoxycholate reversibly inhibited the enzyme(s); removal of detergent restored activity and resulted in the formation of enzymatically active aggregates with a buoyant density of 1.17 g/ml resembling that of membranes. Fractionation of the solubilized membrane components in the presence of 0.2% deoxycholate by either gel filtration or sucrose gradient centrifugation inactivated the enzyme(s) and activity could be restored by the addition of partially purified soybean phospholipids; this allowed some of the physical properties of the enzyme(s) to be determined. 5′-Deiodinating activity of both thyroxine and 3,3′,5′-triiodothyronine was associated with protein(s) with s 20,w of 3.5 S, Stokes' radius of 32 Å, and a calculated molecular weight of 49 900. A partial specific volume of 0.74 cm 3/g was calculated from sedimentation in 2H 2O and H 2O sucrose gradients. Phospholipid reactivation of lipid-depleted enzyme preparations was concentration-dependent, with near maximal restoration when sufficient phospholipid was added to restore the phospholipid:protein ratio to that of the renal microsomes. The enzyme protein(s) catalyzing the 5′-deiodination of thyroxine and of 3,3′,5′-triiodothyronine could not be resolved by sedimentation or molecular sieving and showed similar behavior toward deoxycholate solubilization and phospholipid reconstitution.

The K +- and HCO 3−-stimulated phosphatase activities in renal microsomes of rats

The K +-stimulated phosphatase activity of microsomes from rat kidney was not inhibited by l-phenylalanine, but the HCO 3 −-stimulated phosphatase activity was markedly inhibited by l-phenylalanine. Valinomycin enhanced the HCO 3 −-stimulated phosphatase activity, but did not enhance the K +-stimulated phosphatase activity. Ouabain did not inhibit the HCO 3 −-stimulated phosphatase activity, but inhibited the K +-stimulated phosphatase activity. The renal K +-stimulated phosphatase activity was suppressed to 40% of the control values by adrenalectomy, but the renal HCO 3 −-stimulated phosphatase activity was little suppressed by adrenalectomy. The renal K +-stimulated phosphatase activity in intact and adrenalectomized rats was found to be significantly elevated, in a manner similar to the elevation of the renal (Na + + K +)-ATPase activity by aldosterone treatment ( P < 0.02).

Effects of diuretics on calcium uptake and release in renal microsomes

A variety of diuretics were tested for their effects on Ca 2+ uptake and release by microsomes isolated from rat kidneys. Mersalyl acid, ethacrynic acid, furosemide and bumetanide inhibited Ca 2+ uptake by microsomes. Cysteine abolished the inhibitory effects of mersalyl acid and ethacrynic acid on Ca 2+ uptake. No appreciable differences in the inhibition were seen between microsomes from kidney cortex and those from medulla. Acetazolamide had no significant effect on Ca 2+ uptake; however. hydrochlorothiazide stimulated Ca 2+ uptake activity. The effects of the diuretics on Ca 2+ uptake by microsomes were entirely on the process of Ca 2+ accumulation in microsomes, since release of Ca 2+ that had been taken up previously was not influenced by any of the diuretics tested. These results provide information concerning the biochemical mechanisms of action of diuretics on Ca 2+ reabsorption in the kidney.

Prostaglandin formation by microsomes of dog kidney prostacyclin is a major prostaglandin of dog renal microsomes

Prostaglandin synthesis from arachidonic acid by dog renal microsomes was studied. Formed prostaglandin and thromboxane were identified and quantified by thin layer radiochromatography and thin layer immunochromatography. Inner medullary microsomes formed predominantly 6-keto PGF 1α at a low substrate/enzyme ratio, whereas 6-keto PGF 1α and PGF 2α were the major products at a high substrate/enzyme ratio. On the other hand, cortical microsomes formed predominantly 6-keto PGF 1α at both high and low substrate/enzyme ratio, followed quantitatively by PGF 2α formation.

Enhanced aryl hydrocarbon hydroxylase activity after interaction between solubilized cytochrome P-448 and microsomes low in endogenous cytochrome P-450

The effects of cumene hydroperoxide on microsomal mixed-function oxidase components and enzyme activities were determined. In vitro cumene hydroperoxide treatment decreased cytochrome P-450 content, benzphetamine N-demethylase activity and aryl hydrocarbon hydroxylase activity of hepatic and renal microsomes from adult male and female rats, and of hepatic microsomes from fetal rats. Cumene hydroperoxide-treated microsomes, as well as fetal liver and adult renal microsomes, which are naturally low in cytochrome P-450 and mixed-function oxidase activity, were used to incorporate partially purified hepatic cytochrome P-448 isolated from 2,3,7,8-tetrachlorodibenzo- p-dioxin-pretreated immature male rats. This resulted in an enhanced rate of benzo[ a]pyrene hydroxylation. Aryl hydrocarbon hydroxylase activity was increased 12-, 26-. 31- and 53-fold when 1.0 nmole of partially purified cytochrome P-448 was incubated with fetal liver microsomes, microsomes from kidney cortex of female rats, and cumene hydroperoxide-pretreated hepatic microsomes from female and male rats, respectively. The increased rate of benzo[ a]pyrene hydroxylation was linear with cytochrome P-448 over the range 0.25 to 1.0 nmole. Because cumene hydroperoxide-pretreated microsomes from male rat liver and the hepatic and renal microsomes from female rats have a combination of high NADPH-cytochrome c reductase activity and low mixed-function oxidase activity, they are an attractive choice for catalytic studies of the interaction between cytochrome P-448 and microsomes.

Production of thromboxane A 2 by the kidney in glycerol-induced acute renal failure in the rabbit

The hemodynamic alterations occuring in glycerol induced renal failure are controversial. To date no single humoral substance can fully explain the change in renal resistance observed in this hemodynamic model of acute renal failure. To assess the capacity of the rabbit kidney to produce thromboxane A 2, a potent vasoconstrictor, the following experiments were carried out. Rabbits received 14 ml/kg of 50% glycerol subcutaneously 24 hrs before the study. After 24 hrs., the kidneys were removed and perfused ex vivo in superfusion bioassay cascade. Kidneys from rabbits which developed renal failure, as assessed by elevated serum creatinines, released a substance which produced contraction of rabbit aorta (RCS) in response to bradykinin (BK) and angiotensin II. Microsomes prepared from these kidneys when incubated with [ 14C]-arachidonic acid produced a peak of radioactivity which comigrated with thromboxane B 2 on thin layer chromatography and was inhibited by the thromboxane synthetase inhibitor imidazole. Furthermore, an inverse linear relation was found between the BK dose required to release RCS from perfused kidney and the serum creatinine levels. A direct linear relation was found between the percent of TxB 2 produced by renal microsome preparations and the serum creatinine. These studies demonstrate an increased renal capacity of the glycerol-model of acute renal failure to produce TxA 2. The production of TxA 2 a potent vasoconstrictor should therefore be further evaluated as a potential endogenous mediator of the hemodynamic changes occurring in acute renal failure.

Inhibitory Action of Calcium Antagonists on ATP-Dependent Calcium Uptake by the Renal Cortical Microsomes

Effects of Ca2+ antagonists, verapamil and diltiazem, on uptake and release of Ca2+ by microsomes of the renal cortex were studied. Verapamil inhibited the ATP-dependent Ca2+ uptake by renal microsomes. Addition of 0.8 mM verapamil was required to produce 50% inhibition of the ATP-dependent Ca2+ uptake. Diltiazem also depressed the Ca2+ uptake of the microsomes. The inhibitory effect of both drugs on the Ca2+ uptake was not due to an increased permeability for Ca2+, since release of Ca2+ from the microsomes was not significantly affected by either drug. It is proposed that verapamil and diltiazem inhibit Ca2+ transport by interfering with an active process of Ca2+ accumulation in microsomes of the renal cortex.

Inhibitory action of calcium antagonists on ATP-dependent calcium uptake by the renal cortical microsomes.

Effects of Ca2+ antagonists, verapamil and diltiazem, on uptake and release of Ca2+ by microsomes of the renal cortex were studied. Verapamil inhibited the ATP-dependent Ca2+ uptake by renal microsomes. Addition of 0.8 mM verapamil was required to produce 50% inhibition of the ATP-dependent CA2+ uptake. Diltiazem also depressed the Ca2+ uptake was not due to an increased permeability for Ca2+, since release of Ca2+ from the microsomes was not significantly affected by either drug. It is proposed that verapamil and diltiazem inhibit Ca2+ transport by interfering with an active process of Ca2+ accumulation in microsomes of the renal cortex.

333 - Prostaglandin formation by microsomes of dog kidney Prostacyclin is a major prostaglandin of dog renal microsomes.

Abstract Prostaglandin synthesis from arachidonic acid by dog renal microsomes was studied. Formed prostaglandin and thromboxane were identified and quantified by thin layer radiochromatography and thin layer immunochromatography. Inner medullary microsomes formed predominantly 6-keto PGF1α at a low substrate/enzyme ratio, whereas 6-keto PGF1α and PGF2α were the major products at a high substrate/enzyme ratio. On the other hand, cortical microsomes formed predominantly 6-keto PGF1α at both high and low substrate/enzyme ratio, followed quantitatively by PGF2α formation.

Renal cytochrome P-450's—Electrophoretic and electron paramagnetic resonance studies

The cytochrome P-450' s of the microsomal mixed function oxidase systems from the rabbit renal cortex, outer medulla, inner medulla, and the liver were compared. Sodium dodecyl sulfate-(SDS) gel electrophoresis and electron paramagnetic resonance (EPR) studies detected cytochrome P-450 proteins in the liver, renal cortex, and outer medulla but not the inner medulla of normal animals. Two cytochrome P-450 peptides, which had molecular weights of 54,500 and 58,900 and which comigrated with known hepatic cytochrome P-450' s on SDS gels, were identified in the cortex and outer medulla. Treatment of animals with 3-methylcholanthrene (MC) enhanced the 54,500 and 58,900 peptides in the liver and cortex but produced little change in outer medulla. MC treatment induced faint cytochrome P-450 bands in the inner medulla. The EPR studies detected low spin heme iron absorption lines at g = 2.42, 2.26, and 1.92 in liver, cortex, and outer medulla from untreated animals. The amplitude of the low spin absorption lines was increased by ethanol, a reverse type I compound, and reduced by chloroform, a type I compound, in these tissues. MC treatment increased the amplitude of the heme absorption lines in these tissues, and it induced a barely detectable heme spectrum in the inner medulla. Differences in exogenous substrate binding between hepatic and renal microsomes from MC-treated animals were detected by EPR and optical difference spectroscopy. Acetone, 1-butanol, and 2-propanol gave evidence of binding to the hepatic cytochrome P-450' s but no evidence of binding to renal cortical microsomes. These results, along with previous enzymatic studies, suggest that the liver and each area of the kidney contain different substrate specificities and pathways for the metabolism of organic compounds.

Reversible inhibition of renal microsome calcium pump by furosemide

Renal microsomes have been shown to have an energy-dependent calcium pump activity. We now demonstrate that three diuretic compounds inhibit this calcium pump in vitro . Characterization of furosemide inhibition demonstrates that this agent is a reversible, non-competitive inhibitor of the renal microsome calcium pump. Furosemide, under similar conditions does not inhibit Na/K-ATPase activity in the renal microsome fraction. Furosemide may be useful to define function of the microsomal calcium pump in non-muscle cells.

Chloroform toxicity in the mouse: Role of genetic factors and steroids

DBA/2J male mice are more sensitive to the 10-day lethal effect of chloroform (CHCl3) than are C57BL/6J males, whereas B6D2F1/J mice are intermediate. This relative order of sensitivity is preserved following sublethal doses of CHCl3 in regard to radiolabel accumulation into subcellular biochemical fractions and renal, but not hepatic, dysfunction. Kidneys from mice of all three genotypes are able to repair tubular damage from CHCl3. Thus genotypic differences in response to the toxic effects of CHCl3 are manifestations of differences in renal, rather than hepatic, responses or the ability to repair renal damage. Prior to toxic exposure to chloroform covalent binding of 14CHCl3 to renal microsomes was greater in DBA than in C57BL mice. Phenobarbital pretreatment enhanced covalent binding by renal microsomes from DBA, but not from C57BL mice. Testosterone sensitizes kidneys of both male and female DBA/2J and C57BL/6J mice in a dose-dependent fashion to the toxic effects of CHCl3. The androgenic progestin, medroxyprogesterone acetate, invokes similar responses. These effects are probably mediated in the kidneys by the androgen receptor. Progesterone and hydrocortisone sensitize kidneys to CHCl3 in DBA/2J males, but not in females, nor in C57BL/6J mice of either sex.

Cadmium-induced tissue specific changes in drug biotransformation rates in rats

Cadmium was administered into male rats in drinking water as cadmium chloride at a concentration equivalent to 250 ppm of cadmium for 2 and 8 weeks. The cadmium concentration in the liver microsomes was 0.85 +/- 0.11 microgram/g (wet wt) and in the supernatant 29.6 +/- 1.1 microgram/g and in the renal microsomes 1.30 +/- 0.30 microgram/g (w.wt) and in the supernatant 24.4 +/- 3.2 microgram/g after 8 weeks. In the intestinal postmitochondrial supernatant fraction the cadmium concentration was 14.2 +/- 1.0 microgram/g (wet wt) after 8 weeks administration. There was a slight increase in the hepatic cytochrome P-450 level, no changes in the hepatic p-nitroanisole O-demethylase and NADPH cytochrome c reductase activities and a clearcut induction in the hepatic aryl hydrocarbon hydroxylase activity after 8 weeks cadmium exposure. Renal activities followed mainly those of the liver. No changes were found in the hepatic UDPglucuronosyltransferase activity and a slight activation was present in the renal activity. The intestinal activities were markedly depressed after cadmium exposure. The results suggest that cadmium administration changes the drug biotransformation rates differently in various tissues.

Parathyroid hormone induced stimulation of calcium uptake by renal microsomes

Cortical and papillary microsomes prepared from feline kidneys perfused with parathyroid hormone (PTH) showed an enhanced ability to accumulate calcium (Ca +2). PTH was unable to stimulate Ca +2 uptake into microsomes prepared from outer medulla. These data suggest that renal microsomes may be a valid model system for studying the action of PTH on Ca +2 transport in the kidney.

Renal actions of prostacyclin

A NEW and important member of the arachidonate–thromboxane–prostaglandin system, prostacyclin (PGI2), has recently been isolated1–3. This bicyclic prostaglandin has been shown to be synthesised from the prostaglandin endoperoxides PGG2 and PGH2, by arteries, veins, heart and other tissues in several species, including man1–7. PGI2 is a powerful inhibitor of platelet aggregation and relaxes arterial strips1,2,4,5. It is 30 times more potent than PGE1 as an inhibitor of platelet aggregation, and in the intact dog promotes a dramatic reduction in systemic blood pressure1,2,8,9. It has also been suggested that PGI2 is a potent coronary vasodilator and anti-atherogenic compound10–12, and that it plays an essential part in the homeostasis of the cardiovascular system1,2,4,5,11,12. The kidneys receive a large portion of the cardiac output and have an extensive vascular tree, and so PGI2 may be extremely significant in regulating kidney function. Human and rabbit renal cortical microsomes have been found to convert PGG2 to PGI2 (ref. 13), but nothing is known of the renal effects of PGI2. Therefore, this study was designed to evaluate the effects and significance of an intrarenal infusion of PGI2 on renal haemodynamics, urinary function and renal vein plasma renin activity, and to compare these effects with those produced by PGE2 and PGD2, two prostaglandins known to be synthesised by the kidney and to affect renal function.

Renal prostaglandin synthesis in the spontaneously hypertensive rat.

The precise role of the kidney in spontaneous experimental hypertension is unknown. We have analyzed the rates of renal prostaglandin synthesis by utilizing a spontaneously hypertensive rat model. The synthetic rate of prostaglandin E2, prostaglandin F2alpha, and prostaglandin A2-like products was measured in vitro with renal microsomes. In the rabbit and rat there is a steep gradient of microsomal prostaglandin synthetase from papilla to cortex with highest activities in the papilla. Comparison of the activity of prostaglandin synthetase in medullary microsomes form normotensive and hypertensive rats showed accelerated synthesis in the spontaneously hypertensive rat. These differences appeared after several months of age, were statistically significant from 3 mo of age and, on the average, represented at least a twofold increase of in vitro activity. All classes of prostaglandins were involved with increased synthesis of prostaglandin E2, prostaglandin F2alpha and prostaglandin A2-like material. These data reenforce and extend previous work showing alterations of granularity and presumably prostaglandin synthesis in renal medullary intersitital cells in various experimental hypertensions. We also measured renal tissue content of prostaglandin E and prostaglandin A-prostaglandin B by radioimmunoassay. Swift and careful handling of the tissue was necessary to avoid extensive postmortem synthesis of prostaglandins. In rapidly-frozen medullary tissue only prostaglandin E was detectable in concentrations ranging from 10 to 200 pg/mg tissue. No significant differences were found in the medullary content of prostaglandin E in the control and hypertensive rats despite the increased rates of enzymatic synthesis. We conclude that renal prostaglandin synthesis is increased in renal medullary microsomes obtained from spontaneously hypertensive rat. This apparently occurs in response to the progressive development of hypertension since young animals did not show an increase Renal tissue prostaglandin E content did not increase and therefore appears to be a poor index of enhanced prostaglandin synthesis.

Stimulation of hepatic cytochrome b 5-mediated lipid desaturation by renal microsomes

Although microsomes prepared from rat kidney cortex contained significant concentrations of both NADH cytochrome b 5 reductase and cytochrome b 5, they did not catalyze cytochrome b 5-dependent Δ 9 oxidative lipid desaturation. However, incubation of kidney microsomes in the presence of control liver microsomes resulted in a two-fold increase in fatty acid desaturase activity over that seen with liver microsomes alone. Addition of kidney microsomes to liver microsomes prepared from animals maintained on a fat free diet resulted in an increased desaturase activity which was twice that seen with the control liver preparation. Kidney microsomes alone did not catalyze the cytochrome P-450-dependent N-demethylation of aminopyrine, and in contrast to the desaturate, no increase in demethylase activity was observed when kidney microsomes were added to liver microsomes.

INTRARENAL DISTRIBUTION AND ATPase INHIBITING ACTIVITY OF OUABAIN IN DOGS

Experiments were undertaken to substantiate the hypothesis that the mechanism of the direct effect of ouabain on the renal excretion of electrolytes is the result of inhibition of the transport enzyme, (Na, K)-ATPase. In dogs hydrated with saline, an injection of 3H-ouabain into the unilateral renal artery produced a continuing marked increase in excretion of water and sodium from the kidney, but not from the counter kidney. At maximal diuresis—90 min after ouabain injection, both kidneys were removed to assay microsomal ATPase activity and determine radioactivity distributed in subcellular structures. It was demonstrated that 3H-ouabain was deposited in the microsome fraction obtained from the injected kidney in concentrations ranging from 10−7 to 10−6 M/kg wet weight, and (Na, K)-ATPase activity of this fraction was inhibited as compared with that of the microsomal fraction obtained from control kidneys. Since (Na, K)-ATPase activity of renal microsomes was significantly inhibited in vitro by more than 10−7 M of ouabain, ouabain concentration in microsomes obtained from the injected kidney was considered to be sufficient to inhibit ATPase activity. These findings indicate that ouabain diuresis under the present condition is closely related to direct inhibitory effect of ouabain on (Na, K)-ATPase activity of microsomes in tubular cells.

Energy-dependent calcium uptake activity of microsomes from the aorta of normal and hypertensive rats

Energy-dependent calcium uptake activity of microsomes isolated from the rat aorta has been characterized. The microsomes consist of smooth membrane vesicles which in the presence of Mg · ATP as an energy source continuously sequester calcium over a 60-min period. This calcium uptake is greatly stimulated by oxalate anion which serves as a calcium trapping agent. Unlike the calcium uptake of miltochondria this uptake is not inhibited by sodium azide. Sucrose density gradient analysis of the microsomal calcium uptake suggests that the system is associated with the sarcoplasmic reticulum. In presence of 5 mM Mg · ATP and 20μM calcium approximately 38 nmol of calcium per mg of microsormal protein are taken up in 20 min. In the absence of ATP, less than 2 nmol of calcium per mg of protein are taken up in the first 2 min. with no further uptake of calcium in subsequent time periods. When calcium uptake activity is plotted against calcium or ATP concentration of the medium, half maximal activity is calculated for 24.3 μM calcium and for 1.6 mM ATP. The calcium uptake characteristics of the rat aorta microsomes are compatible with a postulated role in the relaxation of the vascular smooth muscle and the provision of an intracellular calcium store for muscle contraction. Aorta microsomes from SHR rats (a genetic strain that is spontaneously hypertensive) have a significantly reduced calcium uptake when compared with the corresponding nonhypertensive control strain. The level of calcium and ATP for half maximal activity of the rat aorta microsomal calcium uptake system is approximately the same in the SHR and the control strain. The rate of release of calcium from rat aorta microsomes is apparently identical in SHR strain and control. The calcium uptake activity of kidney and liver microsomes isolated from the SHR rat appears to be identical to that found in the control strain. Rats were treated with the steroid deoxycorticosterone acetate for ten and thirty days to induce hypertension. After ten days of deoxycorticosterone acetate although hypertension is present, there is no change in calcium uptake activity of aorta microsomes, renal microsomes or renal plasma membranes. After 30 days of deoxycorticosterone acetate treatment calcium uptake activity of renal microsomes is reduced. A variable decrease in calcium uptake activity is observed with aorta microsomes. Renal plasma membrane calcium uptake remains unchanged.

Effects of testosterone mediated or modulated by pituitary factors.

Adult rats of both sexes were either gonadectomized or hypophysectomized and gonadectomized. Three to eight weeks later they were treated for 14 consecutive days with oil or with 75 or 200 mug testosterone propionate (TP) per 100 g body weight. The animals were killed and for each sex the gonadectomized animals were compared with the hypophysectomized-gonadectomized animals as far as their NADPH- and NADH-dependent 3alpha-hydroxy-steroid dehydrogenases (3alpha-HSD) in renal microsomes, transcortin levels in serum and five organ weights relative to total body weight were concerned. For two of the latter, i.e. the relative kidney and prostatic weights, no significant differences were found. Transcortin levels, relative adrenal weights and renal NADPH-dependent 3alpha-HSD activities were higher in oil-treated gonadectomized animals than in oil-treated hypophysectomized-gonadectomized animals. The opposite was found for the relative weights of uterus and seminal vesicles and renal NADH-dependent 3alpha-HSD activities. These differences between gonadectomized and hypophysectomized-gonadectomized animals disappeared after TP treatment as far as transcortin levels were concerned but remained for the five other parameters. After gonadectomy sexual differences subsisted for all parameters studied. But whereas intact male rats had higher NADH-dependent 3alpha-HSD activities than female rats the opposite was found after gonadectomy. After gonadectomy plus hypophysectomy the between sex differences disappeared as far as transcortin levels were concerned but remained in the other parameters studied.