Garlic ameliorates gentamicin nephrotoxicity: relation to antioxidant enzymes.

Reactive oxygen species are involved in gentamicin (GM) nephrotoxicity, and garlic is effective in preventing or ameliorating oxidative stress. Therefore, the effect of garlic on GM nephrotoxicity was investigated in this work. Four groups of rats were studied: (i) fed normal diet (CT), (ii) treated with GM (GM), (iii) fed 2% garlic diet (GA), and (iv) treated with GM and 2% garlic diet (GM + GA). Rats were placed in metabolic cages and GM nephrotoxicity was induced by injections of GM (75 mg/kg every 12 h) for 6 d. Lipoperoxidation and enzyme determinations were made in renal cortex on day 7. GM nephrotoxicity was made evident on day 7 by (i) tubular histological damage, (ii) enhanced BUN and urinary excretion of N-acetyl-beta-D-glucosaminidase, and (iii) decreased creatinine clearance. These alterations were prevented or ameliorated in GM + GA group. The rise in lipoperoxidation and the decrease in Mn-SOD and glutathione peroxidase (GPx) activities observed in the GM group, were prevented in the GM + GA group. Cu, Zn-SOD activity and Mn-SOD and Cu,Zn-SOD content did not change. CAT activity and content decreased in the GM, GA, and GM + GA groups. CAT mRNA levels decreased in the GM group. The protective effect of garlic is associated with the prevention of the decrease of Mn-SOD and GPx activities and with the rise of lipoperoxidation in renal cortex.

Pertussis toxin potentiates anesthesia-induced renin secretion.

The plasma renin concentration was measured after anesthesia in control and in pertussis toxin-treated rats. The anesthetics increased renin secretion and this effect was markedly magnified in rats treated with pertussis toxin. Propranolol partially blocked the increase in plasma renin concentration produced by anesthetics. It is concluded that the adrenergic system is involved in this effect and that pertussis toxin magnifies it by potentiating the beta-adrenergic action.

Angiotensin I-converting enzyme activity in puromycin aminonucleoside-nephrotic syndrome.

Puromycin aminonucleoside (PAN)-nephrotic rats have high serum angiotensin I-converting enzyme (ACE) activity. We studied ACE activity in serum, urine, and tissues from PAN-nephrotic rats on days 2, 6, 11, and 16 after PAN injection. Proteinuria and hypoproteinemia were evident on days 6 and 11. Though significantly decreased, proteinuria was still evident on day 16. Serum ACE activity increased on days 2, 6, and 11. Urinary ACE activity became evident on days 6, 11, and 16 and correlated positively with proteinuria, suggesting that the source of urine ACE is the blood serum. ACE activity increased in testis on days 2 and 6, in lungs and aorta on days 6 and 11, in adrenal glands and small intestine on day 11, and in kidney on days 11 and 16. Heart ACE activity decreased on days 2 and 6, and increased on day 16; brain ACE activity decreased on day 6 and increased on day 11. These data implicate that changes in tissue ACE content may contribute to elevate serum ACE in PAN-nephrotic rats.

Effect of pertussis toxin on the adrenergic regulation of plasma renin activity.

Basal plasma renin activity (PRA) was not modified by pertussis toxin administration. On the contrary, the modulation of PRA by adrenergic amines was markedly affected by the toxin. Administration of epinephrine did not modified PRA in the controls but markedly increased it in toxin-treated rats. This effect of epinephrine was reproduced in control rats when yohimbine was given before the catecholamine. Clonidine decreased PRA to a much more significant extent in control rats than in animals treated with the toxin. Isoproterenol stimulated PRA to a greater level in toxin-treated rats. Our data indicates that pertussis toxin blocks the alpha 2-adrenergic modulation of renin release and magnifies the ability of beta adrenergic activation to stimulate PRA.

[Renin: structure and expression regulation of the gene, biosynthesis, and cellular pathways of secretion]. / Renina: estructura y regulación de la expresión del gen, biosíntesis y vías celulares de secreción.

The renin angiotensin system plays a major role in the control of blood pressure and electrolyte balance. It consists of a cascade of proteolytic cleavages leading to the biologically active angiotensin II (AII). Renin acts on angiotensinogen to yield angiotensin I (AI). AI is a prohormone and must be cleaved to the octapeptide AII by the action of the angiotensin I converting enzyme. Application of recombinant DNA technology has made possible the cloning of the renin gene and its cDNA which has provided newer insights into the regulation of renin gene expression, biosynthesis, and secretion. The information gained from such molecular biology techniques may contribute importantly to the efforts in the development of an effective renin inhibitor for the treatment of hypertension. The mouse and rat renin gene contains nine exons separated by eight intervening sequences, in contrast the human renin gene contains ten exons separated by nine introns. However, the renin gene of the three species spans 12 kb approximately. In its 5' flanking region, major control elements are present which include promotors and enhancers as well as regulatory elements such as estrogen and glucocorticoid receptor sites, and cAMP induction sequences. The combined action of these elements will result in tissue specific expression and regulation of the gene. In addition to the control at the gene expression level, renin is also regulated at the post-translational and secretory levels. The translational product of renin mRNA is preprorenin, which is cotranslationally cleaved to prorenin, an inactive precursor of renin. The majority of the new synthesized human prorenin is constitutively secreted. However, prorenin is also processed intracellularly and converted to the mature single chain active renin which is stored in secretory granules. Active renin is released by a regulated mechanism which can be stimulated by cAMP and other secretagogues. Studies are under way to examine the responses of renin gene expression, biosynthesis and secretion to various physiological conditions and to determine if there are alterations in the structure and expression of the renin gene that may be involved in the development of clinical and experimental hypertension.

[Structure and regulation of the expression of the angiotensinogen gene]. / Estructura y regulación de la expresión del gen de angiotensinógeno.

Angiotensinogen is a glycoprotein synthesized mainly in hepatocytes and secreted into the circulation. Angiotensinogen is cleaved by the enzyme renin to produce angiotensin I, which is further converted into a vasoconstricting peptide, angiotensin II, the biologically active peptide of the renin angiotensin system. The concentration of angiotensinogen is rate-limiting in the production of angiotensin I and therefore plays an important role in the regulation of angiotensin II production. The development of recombinant DNA technology has introduced new directions for the study of the angiotensinogen molecule. The human, rat and mouse angiotensinogen gene contains five exons interrupted by four intervening sequences and spans 12 kb approximately. In its 5' flanking region multiple regulating elements, as well as the major control elements, are present. The cloning and sequencing of the angiotensinogen cDNA demonstrates the similarity of angiotensinogen to various serine protease inhibitors produced by the liver and was the beginning of studies looking for new physiological roles of angiotensinogen, in addition to the substrate for renin. The circulating levels of angiotensinogen are altered in many different physiological and pathological states. High levels of this protein are seen in hypercorticism, inflammation, pregnancy, and contraceptive therapy, and low levels are associated with adrenal insufficiency and converting enzyme inhibition. These variations are mostly due to modifications of the hepatic biosynthesis under the control of hormonal factors such as glucocorticoid, estrogen, thyroid hormone, insulin and angiotensin II. In addition, it has been found that these changes in the hepatic biosynthesis are due mainly to changes in the angiotensinogen gene expression.

[The current concept of the renin-angiotensin system]. / El concepto actual del sistema renina-angiotensina.

Traditionally, the renin angiotensin system (RAS) has been thought of primarily as an endocrine system that delivers circulating angiotensin II to target tissues. This peptide is a potent vasoconstrictor and a primary stimulus for aldosterone secretion. In addition, angiotensin II has many other targets such as kidney, heart and brain, from which it elicits different specific responses. Numerous studies using pharmacologic or immunologic inhibitors of the system have shown an important role for the circulating RAS in blood pressure and electrolyte as well as fluid homeostasis. Although it acts as a classical circulating endocrine system, there is increasing evidence to show that the RAS may also have an important local autocrine or paracrine role in a variety of tissues since it has been shown that the RAS components are present in all of these tissues. In addition, several investigators have recently demonstrated the expression of renin and angiotensinogen genes in multiple tissues, which strongly suggests that these proteins are locally synthesized. Moreover, it has been demonstrated that tissue RAS is independently regulated from circulating system under different pathological situations such as hypertension. As a result, the concept of the RAS as an endocrine system alone is in question. Locally expressed RAS may be involved with the regulation of individual tissue function independent of the circulating counterpart. However, the importance of these local systems in circulatory control and body volume homeostasis has yet to be defined. It has been proposed that the main function of the circulating RAS is to provide short-term cardiorenal homeostasis. The tonic control (e.g., adrenal and kidney) is influenced by the intrinsic tissue RAS. This new concept provides a broader outlook on the RAS and challenges its traditional endocrine role.

[Intracellular messengers in the regulation of renin secretion]. / Mensajeros intracelulares en la regulación de la secreción de renina.

The intracellular messengers that seem to be involved in renin secretion (RS) from juxtaglomerular cells (JG) are calcium (Ca), cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Unlike the majority of secretory systems, an increase in intracellular Ca concentration and calmodulin and protein kinase C activation inhibit RS. The intracellular Ca concentration in JG cells can be modified if: 1) the normal mechanisms of Ca extrusion of these cells is altered; 2) the calcium output is blocked by lanthanum; 3) the function of the voltage-sensitive Ca-channels is modified; 4) uptake or liberation of Ca from endoplasmic reticulum is modified; 5) plasmatic membrane is bypassed with calcium ionophores such as A 23187. 6) JG cells are stimulated by hormones that increase Ca and activate protein kinase C such as angiotensin II, vasopressin or alpha-1 adrenergic agonists; 7) extracellular Ca concentration increases or decreases. RS is stimulated by dibutyryl cAMP, cAMP phosphodiesterase inhibitors and by hormones and agents that activate adenylate cyclase (beta adrenergic agonists, bradykinin, histamine, forskolin and ethylcarboxamide adenosine). On the contrary, RS is inhibited by hormones and agents that inhibit adenylate cyclase such as: alpha-2 adrenergic agonists, neuropeptide Y, angiotensin II and cyclohexyladenosine. Pertussis toxin increases basal RS, blocks the inhibition by agents and hormones which inhibit adenylate cyclase and potentiate the stimulation produced by beta-adrenergic agonists. In JG cells, atrial natriuretic peptide inhibits RS, increases cGMP and decreases cAMP. The increase in cGMP correlates well with the inhibition of RS.

Pathophysiology of experimental nephrotic syndrome induced by puromycin aminonucleoside in rats. II. In vitro release of renin, angiotensinogen and aldosterone.

In vitro release of renin, angiotensinogen and aldosterone was studied in control (CT) and nephrotic rats. Nephrotic syndrome (NS) was induced by a single injection of puromycin aminonucleoside (PA). The in vitro systems used were: renal cortical slices (RCS), liver slices (LS) and adrenal glands, all incubated in Krebs-Ringer buffer. Renal renin content (RRC) and isoproterenol-induced renin secretion (RS) also were studied. RS, RRC and angiotensinogen release were measured indirectly by radioimmunoassay (RIA) of angiotensin I (ANG I); aldosterone was estimated by direct RIA. Basal RS was not modified in NS: 385 +/- 196 (CT) vs 344 +/- 149 ng ANG I/mg protein/h (NS), p greater than 0.05. Isoproterenol increased RS significantly in both CT and NS groups: 535 +/- (CT) and 685 +/- 231 ng ANG I/mg protein/h (NS) (p less than 0.05 vs. basal RS). RRC was similar in both groups: 2.17 +/- 0.62 (CT) vs 2.05 +/- 0.49 micrograms ANG I/mg protein/h (NS), p greater than 0.05. Angiotensinogen release from LS increased in nephrotic rats from 10 +/- 3.2 (CT) to 12 +/- 1.9 pmoles angiotensinogen I/mg tissue/2h (NS), p less than 0.05. Aldosterone release increased markedly from adrenal glands of rats with NS (1649 +/- 1111 pg aldosterone/mg tissue/h) with respect to control rats (257 +/- 85), p less than 0.05 In vitro studies were performed six days after PA-injection, when nephrotic rats had ascitis, edema, proteinuria, hypoproteinemia, hypercholesterolemia, hypertriglyceridemia, low sodium and aldosterone excretion, low levels of plasma angiotensinogen and high levels of plasma renin and aldosterone.(ABSTRACT TRUNCATED AT 250 WORDS)

Pathophysiology of experimental nephrotic syndrome induced by puromicyn aminonucleoside in rats. III. Effect of captopril, an angiotensin converting enzyme inhibitor, on proteinuria and sodium retention.

The effect of the converting enzyme inhibitor (CEI) (captopril, 50 mg/kg/day) on proteinuria (UProt), urinary aldosterone (UAldoV), plasma renin activity (PRA), plasma renin concentration (PRC), plasma angiotensinogen concentration (PAC), urinary sodium (UNaV), serum total protein, and body weight was studied for 21 days in an experimental nephrotic syndrome (NS) model induced in rats by a single injection (15 mg/100g) of puromycin aminonucleoside (PA). The effect of captopril on control rats without NS was also characterized. In control rats, captopril increased PRC and PRA, and decreased PAC; it had no effect on UNaV, UAldoV, UProt, total serum protein and body weight. In rats with NS, captopril had no effect on sodium retention, hypoproteinemia, and UProt; it abolished the increased UaldoV and favored weight loss. Captopril also rose PRA and PRC, and decreased PAC in PA-nephrotic rats; these changes were similar to those produced by captopril in control rats. The mortality rate was higher in nephrotic rats treated with captopril (37%) than in untreated nephrotic rats (13%). It is concluded that captopril has no beneficial effects on the course on NS induced by PA during the first 21 days, and supports the contention that sodium retention is not related to the renin-angiotensin-aldosterone system activity in these rats.

Pathophysiology of experimental nephrotic syndrome induced by puromycin aminonucleoside in rats. I. The role of proteinuria, hypoproteinemia, and renin-angiotensin-aldosterone system on sodium retention.

The pathophysiology of the nephrotic syndrome (NS), characterized by protenuria, edema, sodium retention and hyperlipidemia, is not clear. We studied the role of some systemic factors on sodium retention in an experimental model of NS. NS was induced in rats by a single subcutaneous injection of puromycin aminonucleoside (PA) (15 mg/100 g); control animals received vehicle. All rats were kept in metabolic cages for 24 days (3 days before and 21 days after PA-injection). Urine was collected daily. Blood samples were obtained every day until day 10, and then every other day up to the end of the study. The rats showed the following alterations after PA injection: a) a rise in serum angiotensin converting enzyme activity (ACEA) and plasma aldosterone (PAldo) at day 1; b) a rise in urinary aldosterone (UAaldoV), azotemia and sodium retention at day 2; c) massive proteinuria (UProt) and decrease in plasma angiotensinogen concentration (PAC) at day 4; d) increases in plasma renin activity (PRA), plasma renin concentration (PRC) and serum creatinine as well as hypoproteinemia, hypercholesterolemia, hypertriglyceridemia, ascitis and edema at day 5; e) increase in urine volume at day 6. PAldo became normal at day 7; urine sodium (UNaV), PRA and PRC at day 8; UAldoV at day 9; serum urea and ACEA at day 10; urinary volume at day 11; PAC, serum total protein and creatinine at day 12. The edema disappeared at day 11. UProt, hypercholesterolemia and hypertriglyceridemia persisted, though they decreased substantially by the end of the study (day 21). Light microscopy studies revealed normal glomerular morphology, but electron microscopy showed fusion of podocytes before proteinuria. These data suggest that: a) sodium retention was not a consequence of proteinuria or hypoproteinemia; b) sodium retention seems non-related to renin secretion, but may be partially mediated by a fall in glomerular filtration rate or by an increased tubular resabsorption secondary to other factors; c) the increase in PAldo, UAldoV and ACEA are non-related to renin secretion: all occurred before PRA rose; d) water retention, increase in PRA and PRC, hypercholesterolemia and hypertriglyceridemia are secondary to the hypoproteinemia.

Circulating levels of active, total and inactive renin (prorenin), angiotensin I and angiotensinogen in carbon tetrachloride-treated rats.

1. Plasma renin activity (PRA), plasma angiotensin I concentration (ANG I), plasma angiotensinogen concentration (PAC) and the plasma levels of active, total and inactive renin (prorenin) were measured in rats with carbon tetrachloride (CCl4)-induced acute renal failure. Rats were treated with a single oral dose of CCl4 (2.5 mL/kg) and killed 1, 2, 3 and 7 days later. 2. On days 1-3 PRA, ANG I and PAC decreased and increased on day 7. Active renin fell on days 2 and 3, total renin (trypsin treatment) augmented on day 1 and diminished on day 3, prorenin and per cent prorenin increased on days 1 and 2. Angiotensin I concentration paralleled PRA and PAC. The CCl4-induced decrease in PRA was secondary to the fall in active renin and in PAC. Total renin augmented as a consequence of the elevation of prorenin. Renal function, evaluated by serum urea, serum creatinine and creatinine clearance, decreased on days 1 and 2 when PRA was low and plasma prorenin was high. 3. These data do not support the involvement of the circulating active renin-angiotensin system (RAS) in the pathophysiology of acute renal failure induced by CCl4, however, increased prorenin levels were associated with the decrease in renal function.

Differential regulation in the expression of hepatic genes in nephrotic and pair-fed rats.

The messenger ribonucleic acid (mRNA) levels of albumin, fibrinogen, alpha 1-acid glycoprotein (pAGP) and transferrin were analyzed in puromycin aminonucleoside (PAN)-treated (nephrotic) and in pair-fed (PF) rats with the Northern and dot blot hybridization techniques. Albumin mRNA levels in nephrotic and PF rats were 2- and 1.5-fold higher, respectively, than in ad-libitum-fed control (C) rats 6 days after PAN treatment. On day 11, this mRNA in PAN-treated rats was 2.5-fold higher than in PF and 4-fold higher than in C rats. A differential expression at the level of specific mRNAs was also detected for fibrinogen, pAGP, and transferrin in nephrotic and PF rats. On day 6, the fibrinogen and transferrin mRNA levels were significantly higher (p < 0.05) in nephrotic than in PF rats. In contrast, pAGP mRNA levels were normal or low in nephrotic rats and increased 2-fold in PF rats. These studies indicate the differential expression of hepatic genes in nephrotic and PF rats and show that albumin gene expression is only partially regulated by diet during the nephrotic stage of the disease.

Kinetic and inhibitory characteristics of serum angiotensin-converting enzyme from nine mammalian species.

1. Serum angiotensin-converting enzyme activities were obtained from nine mammalian species: rat, mouse, horse, sheep, guinea pig, hamster, rabbit, dog and man. 2. Kinetic constants (Km and Vmax) using hippuryl-L-histidyl-L-leucine as substrate and inhibitory constants (I50 and Ki) for captopril were determined for the serum ACE of each species. 3. There were important differences in the kinetic and inhibitory constants (Kms went from 6.6 mM to 1.21 mM for hamster and guinea pig; I50 ranged from 2100 nM to 3 nM for mouse and sheep) as well as differences in enzyme activity of the different species (values varied from 938 to 13 nmol hippuric acid/ml/min for guinea pig and dog serum).

Serum angiotensin converting enzyme activity and plasma renin activity in experimental models of rats.

1. Serum angiotensin converting enzyme activity (ACEA) and plasma renin activity (PRA) were determined in rats under different experimental conditions such as: nephrotic syndrome (NS), bilateral nephrectomy (BN), renovascular hypertension (RH), dehydration (DEH), anaesthesia (AN), low sodium diet (LSD) and high sodium diet (HSD), and injection with propranolol (PRO) and isoprenaline (ISO). 2. PRA increased in LSD, AN, NS, RH, DEH and IPT groups, and decreased in HSD, BN, and PRO groups. Serum ACEA did not change in RH, HSD, IPT, DEH, AN, and PRO groups, increased in NS group, and decreased in LSD and BN groups. 3. Serum ACEA changed in the opposite direction to PRA only in the LSD group. This finding suggests that ACE may limit the full expression of the renin-angiotensin system in the LSD group, but not in the other groups.

Effect of a short-term captopril treatment on serum and tissue angiotensin I-converting enzyme activity from four mammalian species. Differences using diamide in the in vitro assay.

1. Angiotensin I-converting enzyme (ACE) activity was determined in serum and nine tissues from control and captopril-treated rats, mice, guinea pigs and rabbits. 2. ACE activity was determined with and without sample pretreatment with diamide (total and basal activity, respectively). 3. A very different pattern of response to captopril was observed among the different species. 4. There was no relationship between serum ACE activity and the response to captopril. 5. There were important differences in the determinations of total or basal ACE activities. 6. Endogenous ACE inhibitors were found in some tissues from mouse and rabbit.