Adrenal renin expression and its role in ren-2 transgenic rats TGR(mREN2)27

Transgenic rats, termed TGR(mREN2)27, have been generated carrying the mouse ren-2 renin gene. As intended the gene was expressed highly within the adrenal gland offering the opportunity to study the intraadrenal renin-angiotensin system in vivo. Concomitantly with increased adrenal renin activity, the production of adrenocortical steroids are elevated and the regulation of aldosterone production is impaired in TGR(mREN2)27. Furthermore, the transgenic rats develop severe sodium dependent hypertension. Since kidney and plasma renin concentrations and plasma angiotensins are low in the transgenic model, those factors cannot account for the altered regulation of aldosterone production. Instead the experiments with TGR(mREN2)27 demonstrate the functional role of the local adrenal renin-angiotensin system.     

The hypertensive Ren-2 transgenic rat TGR (mREN2)27 in hypertension research. Characteristics and functional aspects

Primary human hypertension is a polygenic disorder. It is the prevalent cause of cardiovascular disease leading to cardiac failure, stroke, chronic renal failure and, ultimately to death. Several genes are involved in cardiovascular control mechanisms and their genetics are complex. Experimental models which are well defined are needed to clarify the role of individual genes. The generation of the hypertensive transgenic rat line TGR (mREN2)27 bearing the murine Ren-2 gene cloned from the DBA/2J mouse strain provides a monogenic model of hypertension in which the genetic basis (the additional renin gene) is known. These rats develop severe hypertension, which reaches 200 mm Hg and higher at 8 weeks of age in the heterozygous animal. Homozygous rats develop even higher blood pressures than heterozygous animals, which is paralleled by a higher mortality rate in homozygous rats. Animals develop pathomorphologic alterations which are characteristic for systemic hypertension. The transgenic rats are characterized by unchanged or even suppressed concentrations of active renin, angiotensin I (ANG I), ANG II, and angiotensinogen compared to transgene-negative littermates. In contrast, plasma levels of inactive renin (prorenin) are much higher in TGR (mREN)27 rats than in control animals. In the kidneys, renin is suppressed, probably mediated through negative feedback inhibition, in other tissues, especially in the adrenal gland, murine Ren-2 mRNA is expressed at very high levels. The cascade of pathophysiologic events which finally lead to hypertension is not fully understood in this rat model. Treatment with ACE inhibitors or angiotensin II receptor antagonists such as losartan is extremely efficient, which could mean that hypertension in this model is mediated through ANG II. Since the the renin-angiotensin system (RAS) in the kidneys is suppressed, other ANG II generating sites must be considered. This favors the concept of extrarenal RASs in this model.     

Increased adrenal renin in transgenic hypertensive rats, TGR(mREN2)27, and its regulation by cAMP, angiotensin II, and calcium

We investigated the effect of the tumor necrosis factor-alpha (TNF alpha) on the differentiation of human adipocyte precursor cells in primary culture. Adipocyte precursors convert into fat cells within 12-16 days in a chemically defined, hormone-supplemented medium. Exposure of cultured preadipocytes to TNF alpha resulted in a dose- and time-dependent decrease in the number of developing fat cells and the activity of glycerol-3-phosphate dehydrogenase (GPDH), an established marker of adipocyte differentiation. A 24-h incubation with TNF alpha at a concentration of 5 nM suppressed GPDH activity by 55% compared to that in control cultures. Continuous exposure of the cells to TNF alpha completely blocked expression of the adipocyte phenotype and GPDH activity. The inhibitory action of TNF alpha was not associated with a change in cell number, as assessed by cell counting. The addition of 5 nM TNF alpha for 24 h to newly developed fat cells caused a rapid reduction of GPDH activity by approximately 50%. A 14-day exposure of differentiated cells to TNF alpha was followed by complete suppression of GPDH and a marked delipidation of the cells, including morphological changes, leading to the development of long spindle-shaped cytoplasmatic extensions. These results clearly demonstrate that TNF alpha inhibits the differentiation of human adipocyte precursor cells and, in addition, promotes the delipidation of mature fat cells. It is suggested that TNF alpha may be involved in the physiological control of human adipose tissue cellularity and function.     

Role of the circulating renin-angiotensin system in the pathogenesis of hypertension in transgenic rats. TGR(mREN2)27

Transgenic rats, termed TGR(mREN2)27, which carry the mouse ren2d renin gene, develop fulminant hypertension. To evaluate the role of the circulating renin-angiotensin system (RAS) in hypertension of TGR(mREN2)27, we determined plasma levels of its components and their regulation by ether-stress. Plasma prorenin was elevated in prehypertensive and in adult heterozygous TGR(mREN2)27 (fourtyfold), when compared with Sprague Dawley (SD) controls, whereas plasma renin concentration (PRC) and angiotensin II were not in SD rats ether anesthesia increased PRC at day (11 a.m.; fivefold), but not at night (11 p.m.). Ether had no effect on PRC in TGR(mREN2)27. In contrast, ether increased plasma corticosterone levels at day and night in both strains to a similar degree. Our data indicate that plasma active renin is not a pathogenetic factor for hypertension in TGR(mREN2)27 and suggest a primary role of circulating prorenin. The lack of stimulation of PRC by ether in TGR(mREN2)27 probably reflects predominant extrarenal origin of renin.     

Progression of renal failure after subtotal nephrectomy in transgenic rats carrying an additional renin gene [TGR(mREN2)27]

Monkey auditory memory was tested with increasing list lengths of 4, 6, 8, and 10 sounds. Five-hundred and twenty environmental sounds of 3-s duration were used. In Experiment 1, the monkeys initiated each list by touching the center speaker. They touched 1 of 2 side speakers to indicate whether a single test sound (presented from both side speakers simultaneously) was or was not in the list. The serial-position functions showed prominent primacy effects (good first-item memory) and recency effects (good last-item memory). Experiment 2 repeated the procedure without the list-initiation response and with a variable intertrial interval. The results of both experiments were similar and are discussed in relation to theories and hypotheses of serial-position effects.     

Differential alteration in vascular structure of resistance arteries isolated from the cerebral and mesenteric vascular beds of transgenic [(mRen-2)27], hypertensive rats

In this study we examined the structural properties of cerebral and mesenteric resistance arteries isolated from normotensive, Sprague-Dawley (SD) rats (mean arterial pressure [MAP], 110 +/- 3 mm Hg) and hypertensive, transgenic (TG) rats (MAP, 167 +/- 4 mm Hg), which express the mouse Ren-2 renin gene. Vessels were set up in a pressure myograph, and ID and vascular wall thickness were determined at increasing intraluminal pressures. Arteries were subsequently pressurized to the MAP of the animal from which they were isolated and were fixed with glutaraldehyde before being embedded in araldite, sectioned, and examined histologically. The middle cerebral artery (MCA) isolated from SD rats and TG rats had similar media cross-sectional areas. There was no difference in MCA diameter at 10 mm Hg in vessels from TG rats compared with SD rats. However, at higher distending pressures, the diameter of the MCA from TG rats was significantly smaller than that of vessels from SD rats. This reduced ID at the higher pressures was a consequence of a decreased distensibility of the MCA from TG rats (as shown by a leftward shift of the stress-strain relationship in arteries from TG rats) and was not caused by an increase in wall thickness. First- and second-order mesenteric resistance arteries isolated from TG rats displayed an increased wall thickness and media content compared with vessels from SD rats. However, this alteration in mesenteric artery structure did not impinge on the ID of arteries from TG rats; there was no difference in the IDs of mesenteric resistance arteries between the two strains at any distending pressure. These observations show that there are distinct regional alterations in vascular structure in hypertensive TG rats expressing the mouse Ren-2 renin gene. Mesenteric resistance arteries isolated from TG rats display signs of vascular growth, although this structural alteration does not produce a reduction in the ID of these arteries per se. In contrast, cerebral arteries from TG rats do not show increased growth but have a reduced vascular distensibility, which results in a smaller ID compared with vessels from SD rats.     

A new model of diabetic nephropathy with progressive renal impairment in the transgenic (mRen-2)27 rat (TGR)

BACKGROUND: The tissue renin-angiotensin system (RAS) may modulate the structural and functional changes that occur in the diabetic kidney. METHODS: Hypertensive transgenic (mREN-2)27 rat (TGR) that exhibit increased tissue renin expression were administered streptozotocin (STZ, diabetic) or citrate buffer (non-diabetic) at six weeks of age, and sacrificed 4 and 12 weeks later. Further groups were treated for 12 weeks post-STZ or vehicle with the angiotensin converting enzyme inhibitor, perindopril. Comparisons were made with 18-week-old non-diabetic and diabetic spontaneously hypertensive rats (SHR). RESULTS: In diabetic TGR, the most florid lesion was seen after 12 weeks of STZ, with kidneys exhibiting vacuolated tubules, hylanized arterioles, medullary fibrosis and necrosis and severe glomerulosclerosis. In contrast, only mild glomerulosclerosis was seen in non-diabetic TGR and diabetic SHR. Glomerular filtration rate was increased after four weeks of diabetes in TGR and 12 weeks of diabetes in SHR, but declined by greater than 50% after 12 weeks of diabetes in TGR. In both TGR and SHR, diabetes increased albuminuria but did not modify systolic blood pressure. Renal renin content increased progressively in diabetic TGR, and this was associated with increased renin immunolabeling in the juxtaglomerular apparatus (JGA) and the appearance of renin in proximal convoluted tubules. In contrast, renal renin content and JGA renin immunolabeling were unchanged in diabetic SHR. Perindopril attenuated renal pathology, improved renal function and abolished proximal tubular renin immunolabeling in diabetic TGR. CONCLUSIONS: This is the first report of a diabetic rodent model developing rapid onset renal impairment. Furthermore, this study suggests a role for an activated renal RAS in the acceleration of diabetic renal disease and confirms the benefit of drugs that inhibit this system.     

Different calcium storage pools in vascular smooth muscle cells from spontaneously hypertensive and normotensive Wistar-Kyoto rats

OBJECTIVE: To evaluate whether the distribution of intracellular free calcium may be impaired in primary hypertension. DESIGN: Cytosolic free calcium and stored calcium were investigated in cultured vascular smooth muscle cells from spontaneously hypertensive rats (SHR). METHODS: The concentrations of intracellular and stored calcium were investigated in cultured vascular smooth muscle cells from spontaneously hypertensive rats aged 6 months from the Münster strain (SHR) and from age-matched normotensive Wistar-Kyoto (WKY) rats. Vascular smooth muscle cells were grown on coverslips, and fluorescence measurements of the intracellular calcium concentration were performed using fura-2. The different effects of thapsigargin, a selective Ca-ATPase inhibitor, and of angiotensin II (Ang II) on the calcium storage pools were investigated. RESULTS: In the absence of external calcium thapsigargin produced a dose-dependent transient increase in the concentration of intracellular calcium in vascular smooth muscle cells. The thapsigargin-induced maximum peak increase in the concentration of intracellular calcium was not significantly different in SHR and WKY rats. After depletion of the thapsigargin-sensitive calcium pools the addition of 100 nmol/l Ang II produced a rise in the concentration of intracellular calcium in vascular smooth muscle cells from SHR and WKY rats. Using vascular smooth muscle cells from the SHR the Ang II-induced increase in the concentration of intracellular calcium was not significantly different in the presence and absence of thapsigargin, indicating that the calcium pools depleted by thapsigargin and Ang II do not overlap significantly in vascular smooth muscle cells from SHR. In contrast, in the WKY rats the response to Ang II was significantly diminished after depletion of the thapsigargin-sensitive pool. When Ang II and thapsigargin were administered in the reverse order, i.e. Ang II before thapsigargin, the thapsigargin response was diminished in the WKY rats but not in the SHR. CONCLUSION: SHR differ from WKY rats in having vascular smooth muscle cells that contain thapsigargin-sensitive calcium storage pools that are distinct from the Ang II-sensitive calcium pools.     

Renal vascular responses to angiotensin II in conscious spontaneously hypertensive and normotensive rats

It has been postulated that exaggerated renal sensitivity to angiotensin II may be involved in the development and maintenance of hypertension in the spontaneously hypertensive rat (SHR). The purpose of this study was to compare the renal vascular responses to short-term angiotensin II infusions (50 ng/kg/min, i.v.) in conscious SHRs and Wistar-Kyoto (WKY) rats. Renal cortical blood flow was measured in conscious rats by using quantitative renal perfusion imaging by magnetic resonance, and blood pressure was measured by an indwelling carotid catheter attached to a digital blood pressure analyzer. Renal vascular responses to angiotensin II were similar in control SHRs and WKY rats. Pretreatment with captopril to block endogenous production of angiotensin II significantly augmented the renal vascular response to exogenous angiotensin II in the SHRs but not in the WKY rats. The renal vascular responses to angiotensin II were significantly greater in captopril-pretreated SHRs than in WKY rats (cortical blood flow decreased by 1.66 +/- 0.13 ml/min/g cortex in WKY rats compared with 2.15 +/- 0.14 ml/min/g cortex in SHR; cortical vascular resistance increased by 10.5 +/- 1.4 mm Hg/ml/min/g cortex in WKY rats compared with 15.6 +/- 1.7 mm Hg/ml/min/g cortex in SHRs). Responses to angiotensin II were completely blocked in both strains by pretreatment with the angiotensin II AT1-receptor antagonist losartan. Results from this study in conscious rats confirm previous findings in anesthetized rats that (a) the short-term pressor and renal vascular responses to angiotensin II are mediated by the AT1 receptor in both SHRs and WKY rats, and (b) the renal vascular responses to angiotensin II are enhanced in SHRs compared with WKY rats when endogenous production of angiotensin II is inhibited by captopril pretreatment.     

Enhanced involvement of endothelin in the haemodynamic sequelae of endotoxaemia in conscious, hypertensive, transgenic ((mRen-2)27) rats

1. Age-matched (3-4 months old) male, heterozygous, hypertensive, transgenic ((mRen-2)27) rats (abbreviated to TG rats) and the normotensive control animals (homozygous, Hannover Sprague-Dawley rats (abbreviated to SD rats), were chronically instrumented for the assessment of regional haemodynamic responses to continuous lipopolysaccharide (LPS) infusion (150 microg kg(-1) h(-1), i.v.) 2. The early (1-2 h) hypotension in SD rats (-11+/-3 mmHg; n=7) was significantly less than that in TG rats (-35+/-3 mmHg; n=8), but by 24 h mean arterial blood pressure (MAP) in both strains of rat was not different from the pre-LPS value (SD rats: baseline, 108+/-3 mmHg; 24 h LPS, 112+/-4 mmHg; TG rats: baseline, 171+/-2 mmHg; 24 h LPS, 169+/-3 mmHg). At this stage in the SD rats there was a renal vasodilatation (delta vascular conductance, 29+/-10 [kHz mmHg(-1)]10(3)) but not in TG rats (delta vascular conductance 2+/-3[kHz mmHg(-1)]10(3)). 3. Co-infusion of LPS and the non-selective endothelin receptor antagonist, SB 209670 (600 microg kg(-1) bolus, 600 microg kg(-1) h(-1)) between 24 and 31 h in SD rats caused a fall in MAP of 16+/-2 mmHg accompanied by hindquarters vasodilatation (delta vascular conductance 11+/-3 (kHz mmHg(-1))10(3)). In TG rats, under the same conditions, the fall in MAP was -60+/-6 mmHg, and there were renal, mesenteric and hindquarters vasodilatations (delta vascular conductance, 23+/-5, 32+/-7, and 14+/-4 (kHz mmHg(-1))10(3), respectively). All effects, except the hindquarters vasodilatation, were greater in TG than in SD rats. 4. In TG rats infused with LPS alone for 31 h, between 24 and 31 h the fall in MAP was -17+/-4 mmHg, and the changes in renal, mesenteric and hindquarters vascular conductances were 5+/-3, -4+/-5, and 12+/-4 (kHz mmHg(-1)10(3), respectively. 5. Administration of the angiotensin (AT1)-receptor antagonist, losartan (10 mg kg(-1), i.v.) following co-infusion of LPS and SB 209670 between 24 and 31 h caused similar falls in MAP in SD and TG rats (-12+/-3 and -14+/-4 mmHg, respectively). 6. These results, together with previous findings, are consistent with a relative enhancement of the contribution of endothelin to the maintenance of cardiovascular status in endotoxaemic TG rats, particularly through a mesenteric vasoconstrictor action.     

Concentration-dependent effects of insulin on Ca2+ influx in vascular smooth muscle cells of normotensive and spontaneously hypertensive rats

The levels of CD26 expression, their capacity to induce protein tyrosine phosphorylation and their functional implication in natural killer (NK) cytolysis have been studied. It was found that only a small fraction (12-15%) of peripheral NK cells expresses CD26 compared with the high expression (99%) found in NK clones. The protein tyrosine phosphorylation mediated by means of CD26 activation was studied in NK cells treated with the anti-CD26 MoAb 134-2C2, and two new proteins of 50 and 21 kDa appeared phosphorylated in tyrosine residues. To study the influence of CD26 antigen in NK lysis, we analysed the lytic capacity of NK cells stimulated with different anti-CD26 MoAbs or after separation into CD26+ and CD26- subsets and using K562 as target cells. Under these conditions, no differences were found in the chromium release by the target cells. Redirected lysis through CD16 was also measured by arming the effector cells (CD26+ and CD26-) with anti-CD16 antibody and using K562 as target cells. It was found that CD26- cells showed significantly less CD16-dependent lysis than CD26+ cells. These results indicate that CD26 is related to the CD16-dependent lysis but not to NK cytolysis which may be caused by mediation of protein tyrosine phosphorylation.     

Angiotensin converting enzyme variability in hypertensive and normotensive rats

Recent data have revealed biological and genetic variability in normotensive Wistar-Kyoto rats, which are considered to be the most appropriate control strain for spontaneously hypertensive rats. To investigate the possibility that angiotensin converting enzyme activity could be affected by this variability, we measured plasma and tissue (lung, heart, renal cortex, renal medulla, and adrenal gland) angiotensin converting enzyme activity in spontaneously hypertensive rats and normotensive Wistar-Kyoto rats from three commercial suppliers in France: Iffa-Credo, Janvier, and Charles River Laboratories. Angiotensin converting enzyme activity was measured in vitro with a fluorometric assay using carbobenzoxy-Phe-His-Leu as substrate. Angiotensin converting enzyme activity in both rat strains varied considerably from one supplier to another, and therefore, comparisons of spontaneously hypertensive rats and Wistar-Kyoto rats from the different suppliers produced conflicting results. For Wistar-Kyoto rats, angiotensin converting enzyme activity in the plasma, heart, kidney, and adrenal glands was highest in rats from Iffa-Credo and lowest in rats from Charles River. For spontaneously hypertensive rats, angiotensin converting enzyme activity in the plasma and tissues was highest in rats from Janvier, whereas no difference could be observed between rats from Iffa-Credo and Charles River. These data confirm the problem of how to interpret and compare studies that use spontaneously hypertensive and Wistar-Kyoto rat strains.     

Comparative effects of the dual metallopeptidase inhibitor, MDL 100,240 and of enalaprilat on regional and on cardiac haemodynamics in conscious, hypertensive, transgenic ((mRen-2)27) rats

1. Heterozygous, male, hypertensive, transgenic ((mRen-2)27) rats (350-450 g) were instrumented for the measurement of regional or cardiac haemodynamics (n = 16, in both groups). Animals were given continuous i.v. infusions of the angiotensin-converting enzyme inhibitor, enalaprilat, or the dual metallopeptidase inhibitor, MDL 100,240 (both at 3 mg kg-1, 3 mg kg-1 h-1; n = 8 for regional and cardiac haemodynamics), for 32 h. Twenty four hours after the onset of infusion of enalaprilat or MDL 100,240, the bradykinin (B2)-receptor antagonist, Hoe 140 (1 mg kg-1, i.v.), was given and measurements were continued for a further 8 h, to assess any possible involvement of bradykinin. 2. Over the first 8 h of infusion, both enalaprilat and MDL 100,240 had significant antihypertensive effects, accompanied by similar regional vasodilatations. However, the blood pressure lowering effect of MDL 100,240 (-54 +/- 9 mmHg) was greater than that of enalaprilat (-38 +/- 4 mmHg), because the former caused a significantly greater reduction in cardiac index. 3. Between 8-24 h after the onset of infusion, there was a reduction in the effect of enalaprilat on blood pressure, because cardiac index rose, with no further increase in total peripheral conductance. In contrast, the antihypertensive effect of MDL 100,240 persisted, in spite of a recovery in cardiac index, because there was further vasodilatation, particularly in the mesenteric and hindquarters vascular beds. 4. There were no apparent haemodynamic changes associated with the injection of Hoe 140, and over the following 8 h, the difference between the haemodynamic effects of enalaprilat and MDL 100,240 persisted; there was little evidence of suppression of the effects of either drug. 5. These results are more consistent with the antihypertensive effects of enalaprilat or MDL 100,240 in transgenic ((mRen-2)27) rats being due to suppression of angiotensin II production, than due to inhibition of bradykinin degradation. The additional effects of MDL 100,240 may be accounted for by inhibition of the degradation of natriuretic peptides reducing cardiac output, initially, and decreasing vascular tone, subsequently. Alternatively, the additional increase in vascular conductance following treatment with MDL 100,240 may represent an autoregulatory response to the reduced pressure.     

Angiotensin II mediates cell hypertrophy in vascular smooth muscle cultures from hypertensive Ren-2 transgenic rats by an amiloride- and furosemide-sensitive mechanism

Matrix metalloproteinases (MMP) are enzymes responsible for extracellular matrix degradation, a critical component influencing the growth and metastatic potential of cancer. The purpose of this study was to determine the in vitro effects of MMP inhibition on human pancreatic cancer cells and to document its effect on cancer growth in vivo. The effect of MMP inhibition was determined using the MMP inhibitor BB-94 and a moderately differentiated pancreatic cancer cell line (HPAC). In vitro, a dose response curve was generated over 5 days utilizing the MTT [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. In vivo, using an established orthotopic model for pancreatic cancer (LD100 = 80 days), 22 nude mice with orthotopic tumors (30 were implanted) received either BB-94 or vehicle beginning 4 days prior to implantation and continuing to death or sacrifice on Day 70. Mice were weighted weekly. At death/sacrifice, tumors were weighted, volume determined, and metastases/ distant spread documented. In vitro, BB-94 had little effect on HPAC proliferation at 40 ng/ml but achieved progressively greater to near complete inhibition at doses up to 4000 ng/ml while maintaining cell viability. In vivo, BB-94 significantly increased length of survival (69 +/- 0.1 days vs. 56 +/- 3.1 days) and necropsy weight (25.7 +/- 1.67 g vs. 19.8 +/- 1.14 g) while decreasing metastatic rate (1 vs. 20) and tumor size (0.14 +/- 0.02 g vs. 0.65 +/- 0.1 g). MMP inhibition limits HPAC proliferation in a dose-dependent fashion without direct cytotoxic effects in vitro. Mice harboring orthotopic tumors treated with BB-94 demonstrated significant reductions in tumor weight, volume, and metastases which corresponded to increased animal weight and prolonged survival.     

Coronary hemodynamics in aging spontaneously hypertensive and normotensive Wistar-Kyoto rats

Conventionally, the hamstring:quadriceps strength ratio is calculated by dividing the maximal knee flexor (hamstring) moment by the maximal knee extensor (quadriceps) moment measured at identical angular velocity and contraction mode. The agonist-antagonist strength relationship for knee extension and flexion may, however, be better described by the more functional ratios of eccentric hamstring to concentric quadriceps moments (extension), and concentric hamstring to eccentric quadriceps moments (flexion). We compared functional and conventional isokinetic hamstring: quadriceps strength ratios and examined their relation to knee joint angle and joint angular velocity. Peak and angle-specific (50 degrees, 40 degrees, and 30 degrees of knee flexion) moments were determined during maximal concentric and eccentric muscle contractions (10 degrees to 90 degrees of motion; 30 and 240 deg/sec). Across movement speeds and contraction modes the functional ratios for different moments varied between 0.3 and 1.0 (peak and 50 degrees), 0.4 and 1.1 (40 degrees), and 0.4 and 1.4 (30 degrees). In contrast, conventional hamstring:quadriceps ratios were 0.5 to 0.6 based on peak and 50 degrees moments, 0.6 to 0.7 based on 40 degrees moment, and 0.6 to 0.8 based on 30 degrees moment. The functional hamstring:quadriceps ratio for fast knee extension yielded a 1:1 relationship, which increased with extended knee joint position, indicating a significant capacity of the hamstring muscles to provide dynamic knee joint stability in these conditions. The evaluation of knee joint function by use of isokinetic dynamometry should comprise data on functional and conventional hamstring:quadriceps ratios as well as data on absolute muscle strength.     

Regulation of preprotachykinin-A mRNA in genetic hypertensive and normotensive rats

It is well-known that central administration of tachykinins (Tks) inhibit salt intake in rats. Recent studies have shown that conditions that arouse salt appetite, such as adrenalectomy and sodium depletion, induce a decrease in preprotachykinin-A (PPT-A) mRNA in discrete regions of the rat brain, suggesting that reduced levels of PPT-A mRNA in the brain may have a permissive role on the expression of salt appetite. It has also been shown that spontaneously hypertensive rats (SHR) show higher avidity for salty solutions than their normotensive control Wistar-Kyoto (WKY) rats. In this regard, the present study tested whether SHR and WKY rats differ in expression of the gene coding for PPT-A, the precursor for Tks peptides. Using semi-quantitative in situ hybridization histochemistry, we examined the level of PPT-A mRNA in discrete rat brain regions of SHR and WKY rats under no treatment, after 1 or 3 days of Na+ depletion. Levels of PPT-A mRNA were analysed in the olfactory tubercle (Tu), in the lateral olfactory tubercle (LOT), in the dorsal and ventral caudate putamen (d/v CPu), in the medial preoptic area (mPOA), in the bed nucleus of the stria terminalis (BNST), in the habenula (Hb) and in the postero-dorsal part of the amygdala (MePD). Semi-quantitative analysis of silver grains revealed a 27.5% lower expression of the PPT-A mRNA levels in SHR opposite to WKY rats under no treatment in v-CPu, mPOA, BNST and Hb. 1 day of Na+ depletion reduced PPT-A mRNA levels when opposite to Na+-repleted animals in Tu and mPOA in both SHR and WKY rats. On the other hand, when comparing SHR and WKY rats after 1 day of Na+ depletion, a 26% lower level of PPT-A mRNA was detected in Tu and d-CPu of SHR opposite to WKY rats whereas a 14% and an 18% lower level was detected in v-CPu and Hb, respectively. A lower expression of PPT-A mRNA in SHR compared to WKY rats was also found in BNST and MePD, although no statistical significance was detected in these two brain areas. In the last experiment, 3 days of Na+ depletion reduced PPT-A mRNA levels in mPOA while negligibly increased mRNA levels in d-CPu and v-CPu, in BNST, Hb and MePD, both in SHR and WKY rats. Conversely, when making comparisons between the two strains, a 35% lower level of PPT-A mRNA in SHR with respect to WKY rats was found after 3 days of Na+ depletion in d-CPu, v-CPu and mPOA. A lower gene expression, even though not statistically significant, was found in Tu, LOT, MePD. These findings show a consistent difference of PPT-A mRNA levels in discrete regions of the SHR brain opposite to WKY rats and confirm that 1 day of Na+ depletion reduces PPT-A mRNA in discrete brain regions. Since SHR are notoriously more salt-avid than WKY rats and Tks are potent inhibitors of sodium intake, the down-regulation of PPT-A mRNA may contribute to the higher natriophilia and, therefore, to the etiology of the hypertensive disease.     

Salt induces myocardial and renal fibrosis in normotensive and hypertensive rats

BACKGROUND: The detrimental effects of high dietary salt intake may not only involve effects on blood pressure and organ hypertrophy but also lead to tissue fibrosis independently of these factors. METHODS AND RESULTS: The effect of a normal (1%) or high (8%) sodium chloride diet on myocardial and renal fibrosis was assessed by quantitative histomorphometry in spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto rats (WKYs). The effect of salt on transforming growth factor-beta1 (TGF-beta1) gene expression was assessed by Northern blot hybridization. A high-salt diet from 8 to 16 weeks of age resulted in increased blood pressure and left ventricular and renal hypertrophy in both WKYs and SHRs. Marked interstitial fibrosis was demonstrated in the left ventricle (LV), glomeruli, and renal tubules and in intramyocardial arteries and arterioles but not in the right ventricle. The collagen volume fraction increased significantly after high-salt diet in the LV, intramyocardial arteries and arterioles, glomeruli, and peritubular areas in both WKYs and SHRs. In the kidneys, glomerular and peritubular type IV collagen was also increased. There was overexpression of TGF-beta1 mRNA in the LV and kidneys in both rat strains after a high-salt diet (all P<0.001). CONCLUSIONS: High dietary salt led to widespread fibrosis and increased TGF-beta1 in the heart and kidney in normotensive and hypertensive rats. These results suggest a specific effect of dietary salt on fibrosis, possibly via TGF-beta1-dependent pathways, and further suggest that excessive salt intake may be an important direct pathogenic factor for cardiovascular disease.