DR Rats Research Articles

Abstract 835: Left Ventricular Expression of Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1 Serves as a Biomarker of Heart Failure in the Salt-Sensitive Dahl Rat Model of Hypertension

Lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1) was originally identified as an endothelial receptor for oxidized LDL, and now is recognized as a multi-ligand receptor. LOX-1 expression in cardiomyocytes can be induced by oxidative stress and various hormonal stimuli. Activation of the LOX-1 pathway in cardiomyocytes induces apoptosis in vitro and deteriorates ischemia-reperfusion injury in vivo . However the role of LOX-1 in chronic heart failure is unknown. We examined the left ventricular (LV) expression of LOX-1 in a salt-sensitive Dahl (DS) rat model of hypertension. Compared with control salt-resistant Dahl (DR) rats, a high-salt diet, started at the age of 6 weeks, induced marked hypertension and apparent concentric LV hypertrophy on echocardiography in DS rats. The LV hypertrophy was moderate at the age of 11 weeks and marked at 18 weeks. The LV systolic function was preserved at 11 weeks, and decreased at 18 weeks. Quantitative real-time PCR revealed a 4.7-fold increase in LV levels of LOX-1 mRNA in DS rats compared with DR rats at 11 weeks, and a 32-fold increase at 18 weeks. The LV LOX-1 mRNA levels were significantly correlated with the LV end-systolic dimension (R=0.640, p=0.0002), and LV posterior wall thickness (R=0.555, p=0.0022), as well as the LV/body weight ratio (R=0.647, p=0.0002), and systolic blood pressure (R=0.751, p<0.0001). LOX-1 levels were also connected with an increase in LV mRNA levels of heme oxygenase-1 (R=0.692, p<0.0001), a marker of oxidative stress. Importantly, LOX-1 levels were strongly associated with a decrease in the LV ejection fraction (R=0.774, p<0.0001) and an increase in mRNA levels of BNP (R=0.814, p<0.0001), a representative marker of LV wall stress and heart failure. Immunohistochemistry demonstrated LOX-1 expression in cardiomyocytes as well as vessel walls of both DS and DR rat hearts. However, expression in cardiomyocytes was greater in DS than in DR. These findings demonstrate that LV cardiomyocyte expression of LOX-1 is markedly increased in proportion to the extent of heart failure and possibly involved in the deterioration of systolic function in the DS rat model of hypertension.

Differential inflammatory status in rats susceptible or resistant to diet-induced obesity: effects of EPA ethyl ester treatment

Obesity has been associated with a chronic low degree inflammatory response, characterized by an increase of inflammatory adipocytokines like tumoral necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) as well as the synthesis of acute phase reactants such as haptoglobin. To evaluate if impairments in the inflammatory response at the white adipose tissue (WAT) level could be involved in the mechanisms conferring susceptibility or resistance to high-fat diet-induced obesity (DIO). The expression levels of WAT genes and systemic markers related to inflammation were evaluated in two groups of rats fed with a high-fat diet during 15 days that showed either an early susceptibility (DIO) or resistance (DR) to develop obesity. We also tested the efficacy of the eicosapentaenoic (EPA) omega-3 fatty acid treatment (35 days) to potentially counteract the obesity-associated inflammatory features in DIO rats. This trial showed that high-fat diet induces an increase on mRNA levels on TNF-alpha and haptoglobin in DIO animals (P < 0.05), while no significant changes were observed on DR rats. Furthermore, a significant increase in IL-6 mRNA (P < 0.05) was found in both DR and DIO rats. EPA-treatment caused a significant decrease in IL-6 mRNA (P < 0.05), without significant changes in haptoglobin mRNA levels in adipose tissue. An unexpected decrease was observed in haptoglobin serum levels (P < 0.05) in DIO rats, which was reverted to control values in EPA-treated animals. Our data suggest that obesity susceptibility or resistance may depend on the genetic make up related to inflammatory features, and support a role for omega-3 fatty acids in the prevention of obesity-associated inflammation in adipose tissue. In addition, our data do not support the hypothesis that serum haptoglobin is an acute phase protein expected to be positively related to increased adiposity in rats, at least in early and medium stages of DIO.

Angiotensin II type 1 receptor blocker preserves tolerance to ischemia-reperfusion injury in Dahl salt-sensitive rat heart

Oxidative stress is involved in the tolerance to ischemia-reperfusion (I/R) injury. Because angiotensin II type 1 receptor blockers (ARBs) inhibit oxidative stress, there is concern that ARBs abolish the tolerance to I/R injury. Dahl salt-sensitive (DS) hypertensive and salt-resistant (DR) normotensive rats received an antioxidant, 2-mercaptopropionylglycine (MPG), or an ARB, losartan, for 7 days. Losartan and MPG significantly inhibited oxidative stress as determined by tissue malondialdehyde + 4-hydroxynoneal and increased expression of inducible nitric oxide synthase (iNOS) in the DS rat heart. However, losartan but not MPG activated endothelial nitric oxide synthase (eNOS) as assessed by phosphorylation of eNOS on Ser1177. Infarct size after 30-min left coronary artery occlusion followed by 2-h reperfusion was comparable between DS and DR rat hearts. Although MPG and losartan had no effect on infarct size in the DR rat heart, MPG but not losartan significantly increased infarct size in the DS rat heart. A selective iNOS inhibitor, 1400W, increased infarct size in the DS rat heart, but it had no effect on infarct size in the losartan-treated DS rat heart. However, a nonselective NOS inhibitor, Nomega-nitro-l-arginine methyl ester, increased infarct size in the losartan-treated DS rat heart. These results suggest that losartan preserves the tolerance to I/R injury by activating eNOS despite elimination of redox-sensitive upregulation of iNOS and iNOS-dependent cardioprotection in the DS rat heart.

Indoxyl sulphate promotes aortic calcification with expression of osteoblast-specific proteins in hypertensive rats

Stage 5 chronic kidney disease (CKD) is associated with enhanced aortic calcification. The aim of this study was to determine if the administration of indoxyl sulphate (IS), a uraemic toxin, stimulates the progression of aortic calcification. The rat groups consisted of (i) Dahl salt-resistant normotensive rats (DR) with intake of 0.3% salt, (ii) Dahl salt-sensitive hypertensive rats (DS) with intake of 2.0% salt and (iii) Dahl salt-sensitive hypertensive IS-administered rats (DS-IS) with intake of 2.0% salt and 200 mg/kg of IS in water. After 30 weeks, their aortic and kidney tissues were excised for histological and immunohistochemical analyses. Severe vascular calcification was observed by von Kossa staining in the arcuate aorta of all the DS-IS rats, but hardly in DS or DR rats. Immunohistochemistry demonstrated that osteopontin, core binding factor 1 (Cbfal), alkaline phosphatase (ALP), osteocalcin, IS and organic anion transporter (OAT) 3 were colocalized in the cells embedded in the aortic calcification area of DS-IS rats. Wall thickness was significantly increased in arcuate, thoracic and abdominal aortas of DS-IS rats compared with DS and DR rats. DS-IS rats showed significantly increased extent of glomerular hypertrophy, mesangial expansion, Masson's trichrome-positive tubulointerstitial area and glomerular and tubulointerstitial expression of transforming growth factor-ssl as compared with DS and DR rats. IS induced aortic calcification with expression of osteoblast-specific proteins and aortic wall thickening. IS is not only a nephrotoxin but also a vascular toxin, and may contribute to the progression of aortic calcification in stage 5 CKD patients.

Different functional responsibility of the small intestine to high-fat/high-energy diet determined the expression of obesity-prone and obesity-resistant phenotypes in rats

The objective of the present experiment was to assess the involvement of small intestine in expression of susceptibility or resistance to the high-fat/high-energy diet. The investigation was carried out in adult male Sprague-Dawley rats fed either standard laboratory diet (3.2 kcal/g, 9.5 % fat) or high-fat (HF) diet (4.04 kcal/g, 30 % fat) for 4 weeks as well as in HF rats that were retrospectively designated on the bases of their higher or lower weight gain as sensitive (DIO) or resistant (DR) to obesity. Our results revealed in HF group significant increase in energy intake, food efficiency, weight gain and Lee s index of obesity. Moreover, in comparison with controls, a significantly increased duodenal and jejunal alkaline phosphatase (AP) and alpha-glucosidase activity as well as hypertrophy of jejunal mucosa (increased protein/DNA ratio) were observed in HF fed rats. In contrast, intestinal function was inversely related to energy intake or to the development of adiposity in DIO vs. DR rats. The DR rats had significantly greater AP and alpha-glucosidase activity and more pronounced suppression of energy intake than obese DIO rats. It indicates that the increase of enzyme activities and the lowered effectiveness of nutrient absorption might be a significant factor preventing the expression of obesity proneness. This information contributes to a better understanding of a complex interaction between HF diet feeding and small intestinal adaptability, which determines the energy homeostasis and predict the ability to resist or develop obesity in these phenotypes.

Dietary zinc deficiency lowers the proportions of splenic CD90+(Thy-1+) B-cells and late thymic emigrant T-cells in growing rats

Zn-deficient (ZD) rats have a lower proportion of splenic CD90+T-cells which could be due to fewer new T-cells exiting the thymus, defective post-thymic maturation or increased cell death. Post-thymic maturation of splenic lymphocytes and their viability were determined by flow cytometry in weanling rats assigned to ZD ( < 1 mg Zn/kg; ad libitum), diet-restricted (DR; 30 mg Zn/kg; limited to the amount of feed as consumed by ZD rats), marginally Zn-deficient (MZD; 10 mg Zn/kg; ad libitum) or control (30 mg Zn/kg; ad libitum) groups for 3 weeks. ZD rats had a 29 % lower percentage of splenic CD90+T-cells and both ZD and DR rats had a 30 % lower proportion of splenic CD90+B-cells compared with control rats. When the splenic CD90+T-cells were characterised further, there was no difference among the groups in the first two stages of post-thymic development; however, ZD, DR and MZD rats had a 42 % lower proportion of late thymic emigrants (TCRalphabeta+CD90+CD45RC+RT6.1+) compared with control rats. There was no difference among groups in the proportion of splenic CD90+T-cells in the non-viable region; however, ZD rats had a higher proportion of CD90+B-cells in the non-viable region compared with MZD and control animals, suggesting that this phenotype was more susceptible to cell death during deficiency. The lower proportion of splenic CD90+T-cells in ZD rats does not appear to be due to a defect in thymic production or increased cell death in the spleen. Future studies should determine if late thymic emigrants have homed to other peripheral organs.

Reduced capacity for fatty acid oxidation in rats with inherited susceptibility to diet-induced obesity

High-fat, energy-dense diets promote weight gain and obesity in humans and other animals, but the mechanisms underlying such diet-induced obesity remain elusive. To determine whether a reduced capacity to oxidize fat is involved in the etiology of diet-induced obesity, we examined different measures of fatty acid oxidation in rats selectively bred for susceptibility (DIO) or resistance (DR) to dietary obesity before and after they were fed a high-fat diet and became obese. DIO rats eating a low-fat diet oxidized less dietary fatty acid in vivo and had lower levels of plasma ketone bodies during fasting compared with DR rats. Lean DIO rats fed a low-fat diet showed reduced liver messenger RNA expression of CD36, which transports fatty acids across cell membranes, and long-chain acyl-coenzyme A dehydrogenase (ACADL), which catalyzes the first step in the mitochondrial β-oxidation of fatty acids. The deficit in CD36 and ACADL messenger RNA expression was also seen in obese DIO rats that had been eating a high-fat diet and, in addition, was accompanied by reduced expression of liver carnitine palmitoyl transferase I, the enzyme that mediates transport of long-chain fatty acids into mitochondria. No differences were found in the expression of liver enzymes involved in fat synthesis; however, in muscle, DIO rats fed the low-fat, but not high-fat, diet showed greater expression of diacylglycerol O-acyltransferase 1 and lipoprotein lipase than did DR rats. Expression of muscle enzymes involved in fatty acid oxidation was similar in the 2 groups. These findings provide a metabolic mechanism for the development of diet-induced obesity and thus suggest potential targets for intervention strategies to treat or prevent it.

Conditioned place preferences (CPPs) to high-caloric “snack foods” in rat strains genetically prone vs. resistant to diet-induced obesity: Resistance to naltrexone blockade

A previous study in our laboratory using Sprague–Dawley (SD) male rats showed that conditioned place preferences (CPPs) can be learned to two different high-caloric “snack foods” — one high in sugar (Froot Loops cereal: FL) vs. one high in fat (Cheetos: C), and that both preferences were mediated by endogenous opioids. Using the same CPP apparatus and procedures, two genetic sub-strains of SD rats, one selectively bred for diet-induced obesity (DIO) vs. another bred for diet resistance to obesity (DR), were used in this investigation. The experiment determined if (a) CPPs can be created in both strains using the same high-caloric “snack foods” and, (b) if CPPs existed, were they opioid dependent. Four non-deprived groups of eight male rats, half being of each strain, were given 20 min sessions to eat either FL or C in one side of a three-chamber CPP apparatus vs. chow on the opposite side over alternating days of a 20 day period. Each predetermined side had distinctly different environmental cues. Following conditioning, rats were tested during 10 min sessions to see if CPPs existed to the “snack food” trained sides. During conditioning and testing, bodyweights, intakes of foods, and activity were measured. Both FL and C generated strong CPPs that were equivalent in both strains. In contrast to our previous study in the parent strain, doses of 0, 0.50, 1.0, 2.5, and 5.0 mg/kg of the opioid antagonist, naltrexone, had no effect on blocking these CPPs. These results show that (a) DIO and DR rats can learn CPPs (i.e., “exhibit food cravings”) as well as their parent strain after periodic access to high-caloric palatable foods, but imply that (b) some physiological system other than the endogenous opioid system mediates such learning.

AGEs mediated expression and secretion of TNF alpha in rat retinal microglia

Diabetic retinopathy induces an inflammatory response in the retina characterized by an increase in inflammatory cytokines and the activation of microglia. The degree of microglia activation may influence the extent of retina injury following retinal metabolic stress. We have previously shown that DR rats have elevated levels of advanced glycation end products (AGEs) in their blood. We have also suggested that AGEs might be involved in microglial activation and production of tumor necrosis factor α (TNF α). In this study, we attempted to confirm that AGEs induce the release of TNF α from rat retinal microglia using an in vitro microglia culture system, and concurrently to explore the mediating mechanisms. AGEs increased the protein secretion and mRNA expression of TNF α in cultured rat retinal microglia. These effects of AGEs were primarily mediated by reactive oxygen species (ROS). Furthermore, the inhibitors for mitogen-activated protein kinases (MAPK; p38, JNK and ERK 1/2) and nuclear factor-kB (NF-kB) could significantly decrease AGEs-induced TNF α release. AGEs-activated microglia showed an increase of NF-kB p65 nuclear translocation. These observations indicated that pathophysiological levels of AGEs may alter rat retinal microglia function by up-regulating TNF α expression and release via enhanced formation of intracellular ROS. AGEs-induced ROS subsequently activates MAPK (p38, JNK and ERK1/2) and NF-kB.

WITHDRAWN: Sex and strain differences impact learning and motivation in DR and DIO rats

The publisher regrets that this article is an accidental duplication of an article that has already been published, doi: 10.1016/j.appet.2006.03.001 . The duplicate article has therefore been withdrawn.

Differential mRNA expression of cyclic‐AMP response element binding protein and neuronal nitric oxide synthase in the dorsal medulla of salt‐sensitive and salt resistant Dahl rats

The purpose of this study was to examine the mRNA levels of cyclic-AMP response element binding protein (CREB) and neuronal nitric oxide synthase (nNOS) in the dorsal medulla of Dahl salt sensitive (DS) and salt resistant (DR) rats. The rats were fed either a regular salt (0.2% NaCl) or a high salt (8% NaCl) diet for three weeks (n= 5 /group). Mean arterial pressure was increased in DS rats (174±10 [mean±SE]) compared to DR rats on high salt (105±4). The animals were sacrificed by decapitation; brain was immediately removed and kept in RNA Later. Gracile nucleus (GR) and nucleus of tractus solitarius (NTS) regions were isolated by micropunch technique from brain sections. Total RNA was isolated and quantitative real time PCR was carried out. mRNA levels of CREB in the NTS region were increased 1.67-fold in DS rats and 1.6-fold in DR rats with high salt compared with the regular salt (p<0.05). nNOS mRNA increased 2.7-fold in both high salt DS and DR rats compared to the normal salt rats (p=0.09). In the GR regions, high salt tended to increase mRNA levels of CREB and nNOS in both DS and DR rats. The results show that high salt diet increases mRNA levels of CREB and nNOS in the NTS region, possibly in the GR region, of DR and DS rats. We conclude that salt loading induces up- regulation of CREB and nNOS mRNA in the dorsal medulla which may contribute to central regulation of the circulation during salt metabolism. Supported by NIH (AT002478, HL04447, and AT00450) to S. Ma.

Hypertension in Diet‐Induced Obese Rats: Neurohumoral Mechanisms Contributing to Blood Pressure

The goal of this study was to determine the arterial pressure and neurohumoral response to high fat intake in rats genetically bred for sensitivity to fat-induced obesity (DIO) or resistance to the fat stimulus (DR). Rats were obtained from Charles River Laboratories. Mean arterial pressure was measured using radiotelemetry starting at 10 weeks of age. MAP was monitored during a one week control period with rats on normal chow. During the control period, MAP was significantly higher in the DIO animals (DIO: 115±1 mmHg vs. DR: 100±1 mmHg). Body weight increased 221±9 gm in the DIO rats and 176±9 gm in the DR rats over a six week high fat feeding period. After the high fat diet, MAP was still elevated in the DIO rats at approximately the same levels observed during the pre-fat period (DIO: 116±1 mmHg vs. DR: 103±1 mmHg). Acute ganlionic blockade caused a greater fall in MAP in the DIO rats compared to the DR animals (−48±4 mmHg vs. 33±2 mmHg); blood pressure was reduced to the same level in both groups. Blood samples taken after high fat diet revealed significantly higher levels of plasma aldosterone (158.4±18.6 ng/ml vs. 61.4±12.1 ng/ml), renin activity (8.3±0.69 ng/ml/hr vs. 5±0.57 ng/ml/hr), and insulin (2.41±0.21 ng/ml vs.1.73±0.17) in the DIO rats compared to the DR animals. These data suggest that genes predisposing animals to obesity on a high fat diet can cause neurogenic hypertension independent of dietary fat content.

A Novel Mechanism Contributing to Development of Dahl Salt–Sensitive Hypertension

To determine the role of the transient receptor potential vanilloid type 1 (TRPV1) channels in development of hypertension in Dahl salt-sensitive (DS) rats fed a high-salt diet (HS), male DS and Dahl salt-resistant (DR) rats were maintained on a low-salt diet (LS) or HS for 3 weeks. HS significantly increased systolic blood pressure in DS+HS rats compared with DS+LS, DR+HS, and DR+LS rats. Intravenous bolus injection of capsazepine (3 mg/kg), a selective TRPV1 antagonist, significantly increased mean arterial pressure in conscious DR+HS rats compared with DR+LS, DS+/-HS, and DS+/-LS rats. In contrast, capsaicin (10 or 30 microg/kg), a selective TRPV1 agonist, dose-dependently decreased mean arterial pressure in all of the groups with the most profound magnitude in DR+HS rats compared with the other 3 groups. TRPV1 expression in mesenteric resistance arteries and the renal cortex and medulla, calcitonin gene-related peptide levels in dorsal root ganglia, and calcitonin gene-related peptide-positive sensory nerve density in mesenteric resistance arteries were significantly decreased in DS+HS rats compared with DS+LS, DR+HS, and DR+LS rats. Taken together, our data indicate that the TRPV1 receptor is activated and its expression upregulated during HS intake in DR rats, which acts to prevent salt-induced increases in blood pressure. In contrast, TRPV1 expression and function are impaired in DS rats, which renders DS rats sensitive to salt load in terms of blood pressure regulation.

Eplerenone Shows Renoprotective Effect by Reducing LOX-1–Mediated Adhesion Molecule, PKCε-MAPK-p90RSK, and Rho-Kinase Pathway

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) may play an important role in atherosclerosis by inducing leukocyte adhesion molecules, such as intercellular and vascular cell adhesion molecule-1 (intercellular adhesion molecule-1 [ICAM-1], vascular cell adhesion molecule-1 [VCAM-1]). We hypothesized that eplerenone, a novel selective aldosterone blocker, produces inhibition of LOX-1-mediated adhesion molecules, suppresses mitogen-activated protein (MAP) kinase and its downstream effector p90 ribosomal S6 kinase (p90RSK) through the protein kinase Cepsilon (PKCepsilon) pathway, and improves endothelial function by inhibition of Rho-kinase in the renal cortex of Dahl salt-sensitive hypertensive (DS) and salt-resistant (DR) rats. Eplerenone (10, 30, and 100 mg/kg per day) was given from the age of 6 weeks to the left ventricular hypertrophy stage (11 weeks) for 5 weeks. At 11 weeks, expression levels of LOX-1, ICAM-1, VCAM-1, and Rho-kinase were higher in DS rats than in DR rats and were decreased by eplerenone. Similarly, upregulated phosphorylation of PKCepsilon, MAP kinase, and p90RSK in DS rats was also inhibited by eplerenone. In contrast, downregulated endothelial nitric oxide synthase mRNA was increased by eplerenone to a similar degree as after treatment with Y-27632, a selective Rho-kinase inhibitor. Eplerenone administration resulted in significant improvement in glomerulosclerosis (eplerenone 10 mg, -61%; 30 mg, -78%; and 100 mg, -84% versus DS; P<0.01, respectively) and urinary protein (10 mg, -78%; 30 mg, -87%; and 100 mg, -88% versus DS; P<0.01, respectively). These results suggest that the renoprotective effects of eplerenone may be partly caused by inhibition of LOX-1-mediated adhesion molecules and PKCepsilon-MAP kinase-p90RSK pathway, and improvement in endothelial function.

11β-Hydroxysteroid dehydrogenase in the heart of normotensive and hypertensive rats

Corticosteroids have been shown to play a role in cardiac remodeling, with the possibility of a direct effect of overexpression of 11β-hydroxysteroid dehydrogenase (11HSD) isoform 2 at the level of the cardiomyocytes. The aim of this study was to examine cardiac steroid metabolism in hypertensive rats with hearts that are hypertrophied and fibrotic and have structural alterations in the coronary circulation. To assess possible alterations of cardiac steroid metabolism the expression and activity of both isoforms of 11β-hydroxysteroid dehydrogenase (11HSD) were studied in spontaneously hypertensive rats (SHR), their normotensive controls Wistar–Kyoto (WKY), and in Dahl salt-sensitive (DS) and salt-resistant rats (DR) kept on a low- or high-salt diet. Using real-time quantitative RT-PCR and enzyme activity assay we found strain-dependent differences in cardiac metabolism of glucocorticoids. In Dahl rats expression of 11HSD1 and 11HSD2 mRNA was lower in DS than in DR rats and was not influenced by dietary salt intake; 11HSD1 mRNA was expressed at higher level than 11HSD2 mRNA. NADP +-dependent cardiac 11HSD activity showed similar distribution as 11HSD1 mRNA—lower activity in DS than in DR rats and no effect of salt intake. In SHR and WKY strains 11HSD2 mRNA expression was significantly higher in WKY than in SHR but no differences were observed in 11HSD1 mRNA abundance and NADP +-dependent 11HSD activity. These results show that the heart is able to metabolize glucocorticoids and that this metabolism is strain-dependent but do not support the notion of association between cardiac hypertrophy and changes of 11HSD1 and 11HSD2 expression.

Changes of hypothalamic α-MSH and CART peptide expression in diet-induced obese rats

Two hypothalamic peptides, cocaine and amphetamine-regulated transcript (CART) and α-melanocyte-stimulating hormone (α-MSH), recognized as anorexigenic neuropeptides to suppress the feeding behavior, were monitored in rats fed with a high-fat (HIF) diet for 14 weeks. While half of the rats developed obesity (diet-induced obese, DIO), some did not (diet resistant, DR). Compared to the DR rats and the control rats (fed with standard chow), DIO rats were accompanied by a markedly higher energy intake and a decrease in the number of neurons carrying α-MSH and CART peptide in the arcuate nucleus of the hypothalamus. Failure of hypothalamic anorexigenic peptides CART and α-MSH to increase their content in response to HIF diet may play a key role for overly high energy consumption, resulting in obesity.

Angiogenesis and arteriogenesis are increased in pre-hypertensive SHR

Renal NaCl reabsorption is increased in Dahl "salt-sensitive" (DS) rats, due to an increased activity of the Na-K-Cl cotransporter NKCC2. On the other hand, nitric oxide (NO) is an inhibitor of NKCC2 and a deficient nitric oxide synthase (NOS) seems to play an important role in salt-sensitivity of DS rats. Here, we investigated the hypothesis that NKCC2 hyperactivity in DS rats is due to a deficient NOS, via the interactions cyclic GMP (cGMP)/cyclic AMP (cAMP) at the level of the thick ascending Henle's loop (TAL). DS rats DS (males, 250-300 g) and their normotensive controls DR ("salt-resistant") are sacrificed, the kidneys removed and NKCC2 activity is measured in medullary TAL (mTAL) as previously described. Medullary contents of NO are measured with a NitroFlux analyser by heat-reduction of nitrates and nitrites to NO. AMPc levels in mTAL are measured by an EIA immunotest. Neither L-NAME (3 mM), nor L-arginine were able to modify NKCC2 activity in mTAL from DS (pre-hypertensive) or DR rats. Levels of NO in the medullary interstitium and AMPc in mTAL were not significantly different between DS and DR rats. Conversely, in DS rats charged with 2% salt (in the food) during 7 weeks, L-arginine significantly inhibited NKCC2 in DS (35.6 +/- 6.8 vs 25.3 +/- 4.9 nmoles/mg protein/min; p<0.05 non-paired Student's t-test), but not in DR rats. In conclusion, NKCC2 in our mTAL preparation of prehypertensive DS and DR rats is insensitive to L-NAME and L-arginine. This suggests the absence of a functional NOS. NKCC2 hyperactivity of prehypertensive DS is therefore not due to a deficient NOS. This was confirmed by the normal levels of interstitial NO and mTAL cAMP in prehypertensive DS rats. Finally, a salt-load seems to induce NOS expression in mTAL of DS rats. This last observation deserves further investigation.

Increased collecting duct urea transporter expression in Dahl salt-sensitive rats.

Because abnormalities of inner medullary function have been proposed in Dahl salt-sensitive (DS) rats vs. salt-resistant (DR) rats, we performed transporter profiling by semiquantitative immunoblotting to determine whether specific solute transporter abundances are altered in inner medullas of DS rats vs. DR rats. Although none of the expressed Na transporters were upregulated in the inner medullas of DS rats compared with DR rats, there were marked increases in the protein abundances of the collecting duct urea transporters UT-A1 (to 212% of DR) and UT-A3 (to 223% of DR). These differences were confirmed by immunocytochemistry. Quantitative real-time RT-PCR showed higher mRNA abundance in DS rats for both UT-A1 (to 256% of DR) and UT-A3 (to 210% of DR). In isolated, perfused inner medullary collecting ducts, urea permeability was significantly greater in DS rats. Because both UT-A1 and UT-A3 are transcriptionally regulated by glucocorticoids, we measured both plasma corticosterone levels and inner medullary 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) abundances. Although the plasma corticosterone concentrations were not different between DS and DR rats, immunoblotting and immunocytochemistry revealed a marked elevation of 11beta-HSD2 abundance in DS rats. Consistent with the view that an elevated 11beta-HSD2 level is responsible for increased urea transporter expression in the inner medullary collecting duct, administration of the 11beta-HSD2 inhibitor carbenoxolone to DS rats decreased the abundances of UT-A1 and UT-A3 to levels similar to those seen in DR rats.

Mechanisms of increased liver tissue repair and survival in diet-restricted rats treated with equitoxic doses of thioacetamide.

Moderate dietary or caloric restriction (DR) modulates animal physiology in a beneficial fashion. Previously, we have reported an equitoxic dose experiment where liver injury in DR male Sprague-Dawley rats exposed to a low dose of thioacetamide (TA, 50 mg/kg) was similar to that observed in ad libitum fed (AL) rats exposed to a 12-fold higher dose (600 mg/kg). Paradoxically, the AL rats experienced 90% mortality while all of the DR rats, with the same amount of initial bioactivation-mediated liver injury, survived. The protection observed in the DR rats was due to efficient compensatory liver tissue repair, which was delayed and attenuated in the AL rats, leading to progression of liver injury. The objective of the present study was to investigate the molecular mechanisms of the enhanced tissue repair in the DR rats upon equitoxic challenge with TA. Promitogenic mechanisms and mediators such as proinflammatory cytokines (TNF-alpha and IL-6), growth factors (TGF-alpha and HGF), and inducible nitric oxide synthase (iNOS) were estimated over a time course after equitoxic challenge (50 mg/kg to DR vs. 600 mg/kg to AL rats). Except for TNF-alpha, all other molecules were expressed earlier and in greater amount in the DR rats. IL-6 was 10-fold greater and peaked 12 h earlier; HGF also peaked 12 h sooner in the DR rats, when it was 2.5-fold greater than the value in the AL rats. TGF-alpha expression in livers of DR rats increased after TA administration and peaked at 24 h. In the AL rats, it was lower and peaked at 36 h. Diet restriction alone induced iNOS 2-fold in the DR rats and remained elevated until 12 h after TA administration, then declined thereafter. The lower iNOS activity in the AL rats further decreased after TA injection. DR rats exhibited higher apoptosis after thioacetamide administration, which further increased the efficiency of tissue repair. Taken together, these data indicate that even though the liver injury is near equal in AL and DR rats, sluggish signal transduction leads to delayed liver regeneration, progression of liver injury, and death in the AL rats. The equitoxic dose experiment indicates that stimulation of tissue repair is independent of the extent of initial liver injury and is governed by physiology of diet restriction. DR stimulates promitogenic signaling leading to a quick and timely response upon liver injury, arrest of progressive injury on one hand, and recovery from injury on the other, paving the way for survival of the DR rats.

Upregulated promitogenic signaling via cytokines and growth factors: potential mechanism of robust liver tissue repair in calorie-restricted rats upon toxic challenge.

Previously we reported that moderate calorie restriction or diet restriction (DR, calories reduced by 35% for 21 days) in male Sprague-Dawley rats protects from a lethal dose of thioacetamide (TA). DR rats had 70% survival compared with 10% in rats fed ad libitum (AL) because of timely and adequate compensatory liver cell division and tissue repair in the DR rats. Further investigation of the mechanisms indicate that enhanced promitogenic signaling plays a critical role in this stimulated tissue repair. Expression of stimulators of promitogenic signaling interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), hepatocyte growth factor (HGF), transforming growth factor-alpha (TGF-alpha), and epidermal growth factor receptor (EGFR) were studied during liver tissue repair after TA-induced liver injury. Plasma IL-6 was significantly higher in the DR rats, with 6-fold higher expression at 48 h after TA administration. Immunohistochemical localization revealed significantly higher expression of IL-6 in the hepatic sinusoidal endothelium of DR rats. Expression of TGF-alpha and HGF was consistently higher in the livers of DR rats from 36 to 72 h. EGFR, which serves as a receptor for TGF-alpha, was higher in DR rats before TA administration and remained higher till 48 h after TA intoxication. DR-induced 2-fold increase in hepatic iNOS activity is consistent with early cell division in DR rats after TA challenge. These data suggest that the reason behind the higher liver tissue repair after TA-induced hepatotoxicity in DR rats is timely and higher expression of the growth stimulatory cytokines and growth factors. It appears that the physiological effects of DR make the liver cells vigilant and prime the liver tissue promptly for liver regeneration through promitogenic signaling upon toxic challenge.