Effects of separated pair housing of female C57BL/6JRj mice on well-being.

In laboratory animal facilities, it is a common code of practice to house female mice in groups. However, some experimental conditions require to house them individually, even though social isolation may impair their well-being. Therefore, we introduced a separated pair housing system and investigated whether it can refine single housing of adult female C57BL/6JRj mice. Individually ventilated cages (IVC) were divided by perforated transparent walls to separate two mice within a cage. The cage divider allowed visual, acoustic, and olfactory contact between the mice but prevented interindividual body-contact or food sharing. Short- and long-term effects of the separated pair housing system on the well-being of the mice were compared with single and group housing using a range of behavioral and physiological parameters: Nest building behavior was assessed based on the complexity of nests, the burrowing performance was measured by the amount of food pellets removed from a bottle, and trait anxiety-related behavior was tested in the free exploratory paradigm. For the evaluation of the ease of handling, interaction with the experimenter's hand was monitored. Social interaction with unknown conspecifics and locomotor activity were investigated in a test arena. Moreover, body weight and stress hormone (metabolites) were measured in feces and hair. After the mice spent a day under the respective housing conditions, concentrations of fecal corticosterone metabolites were higher in separated pair-housed mice, and they built nests of a higher complexity when compared to single-housed mice. The latter effect was still observable eight weeks later. In week 8, separated pair-housed mice showed less locomotor activity in the social interaction arena compared to mice from the other housing systems, i.e., single and group housing. Regardless of the time of testing, pair housing improved the burrowing performance. Separated pair-housed mice were more difficult to catch than group-housed mice. Hair corticosterone, progesterone, and dehydroepiandrosterone concentrations changed with increasing age independently of the housing system. There were no effects of the housing systems on trait anxiety-related behavior in the free exploratory paradigm, voluntary interaction with the experimenter's hand, and body weight. Overall, the transfer to the separated pair housing system caused short-term stress responses in female C57BL/6JRj mice. Long-term effects of separated pair housing were ambiguous. On one hand, separated pair housing increased nesting and burrowing behavior and may therefore be beneficial compared to single housing. But on the other hand, locomotor activity decreased. The study underlined that the effects of the housing conditions on physiological and behavioral parameters should be considered when analyzing and reporting animal experiments.

Short communication: Is hair cortisol a potential indicator for stress caused by chronic lameness in dairy cows?

The objective of this study was to evaluate hair cortisol concentration as an indicator for stress caused by chronic lameness in dairy cows. Sixty-eight cows were scored for lameness for 4 consecutive weeks. The hair of the tail switch was clipped at the beginning of the study and regrown hair was clipped after 4 wk. Hair samples were analyzed for cortisol concentration. Animals with 2 consecutive locomotion scores ≥3 or with an overall mean score >1.5 were classified as lame. After pair matching lame and nonlame cows, considering days in milk, lactation number, and milk yield, and excluding cows with less than 20 mg hair sample for analysis, 21 lame and 21 nonlame cows were included in the analysis. The mean hair cortisol concentration in this study was 2.32 ± 0.35 pg/mg (mean ± standard deviation). Cortisol concentration from hair regrown in the study period was 2.38 ± 0.95 and 2.26 ± 1.35 pg/mg for lame and nonlame cows (n = 21), respectively; we found no difference in mean cortisol level of primiparous and multiparous cows. Based on these data, hair cortisol concentration was not a useful indicator to differentiate cows with chronic lameness and healthy cows.

Renal outcome in equipotent antihypertensive treatment with telmisartan, ramipril and in combination in SHR-SP rats.

BACKGROUND: The ONTARGET trial revealed an association of ACEI/ARB combination treatment (telmisartan and ramipril) with adverse renal outcome versus respective monotherapy; preclinical evidence regarding renal outcome in ACEI/ARB combination treatment is scarce. METHODS: Spontaneously hypertensive stroke prone rats (SHR-SP) rats on a salt-rich diet were randomly allocated to 4 groups: SHR (untreated, n = 24), SHR + telmisartan (SHR-T, 2.39 +/- 0.69 mg/kg bw; n = 27), SHR + ramipril (SHR-R, 6.28 +/- 3.48 mg/kg bw; n = 27) and combination treatment (SHR-TR, 0.51 +/- 0.14 mg/kg bw; same dose for telmisartan and ramipril; n = 26). Study duration was 12 weeks, blood pressure was assessed weekly and doses were adjusted to maintain equal blood pressure. Finally, blood and urine samples were obtained and kidneys were harvested for histological studies. RESULTS: Blood pressure in untreated rats rose to a maximum of 239 mmHg, whereas in all treatment groups it remained stable between 140 and 150 mmHg. Mortality was 50% in the untreated group, whereas all treatment groups survived completely. Renal function--as indicated by plasma urea and cystatin c--was significantly worse in SHR-TR animals compared to all other groups. With plasma creatinine a similar trend was observed. All treatment options significantly decreased albuminuria. Renal glomerulosclerosis was decreased by monotherapy, whereas combination therapy failed to have a significant effect. Interstitial fibrosis was decreased to a similar extent by all treatment options. CONCLUSIONS: ACEI/ARB combination treatment failed to render significant additional benefits on renal outcome in hypertensive rats when compared to monotherapy. Instead our data indicate that dual RAAS blockade might have an adverse effect on kidney function and histology when compared to monotherapy in salt-loaded SHR-SP.

Renal phenotype of ET-1 transgenic mice is modulated by androgens.

INTRODUCTION: Activation of the endothelin (ET) system promotes inflammation and fibrosis in various tissues including the kidney. Male ET-1 transgenic mice are characterized by chronic kidney inflammation and renal scarring. We hypothesized that this renal phenotype might be modulated by androgens. Thus the aim of our study was to elucidate the impact of gonadectomy in ET-1 transgenic mice on kidney function and morphology. - METHODS: Male ET-1 transgenic mice at the age of 10 weeks were randomly allocated to the following groups: normal ET transgenic mice (ET; n = 17) and ET transgenic mice that underwent castration (ET + cas; n = 12). Study duration was 9 months. Creatinine clearance and protein excretion was monitored. At study end animals were sacrificed and kidneys were harvested for histology/immunhistochemistry. RESULTS: Castration significantly ameliorated glomerulosclerosis in ET-1 transgenic mice (ET glomerulosclerosis-score: 3.0 +/- 0.17 vs ET+cas: 2.4 +/- 0.17; p < 0.05) as well as renal perivascular fibrosis (ET fibrosis-score: 3.0 +/- 0.14 vs ET + cas: 2.2 +/- 0.14; p < 0.05). However, interstitial fibrosis and media/lumen-ratio of renal arteries remained unaffected by castration. Regarding inflammation, castration significantly reduced the number of CD4-positive cells in renal tissue of ET-1 transgenic mice (ET CD4-positive cells/10000 cells: 355 +/- 72 vs ET + cas: 147 +/- 28; p < 0.05). Renal tissue contents of CD8 positive cells as well as of macrophages were not affected by castration. Regarding kidney function castration significantly reduced proteinuria in ET-1 transgenic mice whereas creatinine clearance did not differ between study groups. CONCLUSION: Our study demonstrates that the renal histopathological phenotype in male ET-1 transgenic mice with regard to glomerulosclerosis, proteinuria, perivascular fibrosis and immune cell immigration is ameliorated by castration. We thus conclude that the effects of ET-1 overexpression on renal tissue injury are modulated by androgens.

High-protein nutrition during pregnancy and lactation programs blood pressure, food efficiency, and body weight of the offspring in a sex-dependent manner.

Maternal low-protein diet during pregnancy is a risk factor for cardiovascular disease of the offspring in later life. The impact of high-protein diet during pregnancy on the cardiovascular phenotype of the offspring, however, is still unknown. We examined the influence of a high-protein diet during pregnancy and lactation on the renal, hemodynamic, and metabolic phenotype of the F1 generation. Female Wistar rats were either fed a normal protein diet (20% protein: NP) or an isocaloric high-protein diet (40% protein: HP) throughout pregnancy and lactation. At weaning, the offspring were fed with standard diet, and they were allocated according to sex and maternal diet to four groups: normal-protein male (NPm, n = 25), normal-protein female (NPf, n = 19), high-protein male (HPm, n = 24), high-protein female (HPf, n = 29). During the experiment (22 wk), the animals were characterized by repeated measurement of body weight, food intake, blood pressure, glucose tolerance, energy expenditure, and kidney function. At the end of the study period histomorphological analyses of the kidneys and weight measurement of reproductive fat pads were conducted. There were no differences in birth weight between the study groups. No influence of maternal diet on energy expenditure, glucose tolerance, and plasma lipid levels was detected. Blood pressure and glomerulosclerosis were elevated in male offspring only, whereas female offspring were characterized by an increased food efficiency, higher body weight, and increased fat pads. Our study demonstrates that a high-protein diet during pregnancy and lactation in rats programs blood pressure, food efficiency, and body weight of the offspring in a sex-dependent manner.

PYY3-36 as an anti-obesity drug target.

The neuropeptide Y (NPY)/peptide YY (PYY) system has been implicated in the physiology of obesity for several decades. More recently ignited enormous interest in PYY3-36, an endogenous Y2-receptor agonist, as a promising anti-obesity compound. Despite this interest, there have been remarkably few subsequent reports reproducing or extending the initial findings, while at the same time studies finding no anti-obesity effects have surfaced. Out of 41 different rodent studies conducted (in 16 independent labs worldwide), 33 (83%) were unable to reproduce the reported effects and obtained no change or sometimes increased food intake, despite use of the same experimental conditions (i.e. adaptation protocols, routes of drug administration and doses, rodent strains, diets, drug vendors, light cycles, room temperatures). Among studies by authors in the original study, procedural caveats are reported under which positive effects may be obtained. Currently, data speak against a sustained decrease in food intake, body fat, or body weight gain following PYY3-36 administration and make the previously suggested role of the hypothalamic melanocortin system unlikely as is the existence of PYY deficiency in human obesity. We review the studies that are in the public domain which support or challenge PYY3-36 as a potential anti-obesity target.

Epigallocatechin gallate attenuates diet-induced obesity in mice by decreasing energy absorption and increasing fat oxidation.

OBJECTIVE: To examine the antiobesity effect of epigallocatechin gallate (EGCG), a green tea bioactive polyphenol in a mouse model of diet-induced obesity. METHODS: Obesity was induced in male New Zealand black mice by feeding of a high-fat diet. EGCG purified from green tea (TEAVIGO) was supplemented in the diet (0.5 and 1%). Body composition (quantitative magnetic resonance), food intake, and food digestibility were recorded over a 4-week period. Animals were killed and mRNA levels of uncoupling proteins (UCP1-3), leptin, malic enzyme (ME), stearoyl-CoA desaturase-1 (SCD1), glucokinase (GK), and pyruvate kinase (PK) were analysed in different tissues. Also investigated were acute effects of orally administered EGCG (500 mg/kg) on body temperature, activity (transponders), and energy expenditure (indirect calorimetry). RESULTS: Dietary supplementation of EGCG resulted in a dose-dependent attenuation of body fat accumulation. Food intake was not affected but faeces energy content was slightly increased by EGCG, indicating a reduced food digestibility and thus reduced long-term energy absorption. Leptin and SCD1 gene expression in white fat was reduced but SCD1 and UCP1 expression in brown fat was not changed. In liver, gene expression of SCD1, ME, and GK was reduced and that of UCP2 increased. Acute oral administration of EGCG over 3 days had no effect on body temperature, activity, and energy expenditure, whereas respiratory quotient during night (activity phase) was decreased, supportive of a decreased lipogenesis and increased fat oxidation. CONCLUSIONS: Dietary EGCG attenuated diet-induced body fat accretion in mice. EGCG apparently promoted fat oxidation, but its fat-reducing effect could be entirely explained by its effect in reducing diet digestibility.

Physiology: does gut hormone PYY3-36 decrease food intake in rodents?

Batterham et al. report that the gut peptide hormone PYY3-36 decreases food intake and body-weight gain in rodents, a discovery that has been heralded as potentially offering a new therapy for obesity. However, we have been unable to replicate their results. Although the reasons for this discrepancy remain undetermined, an effective anti-obesity drug ultimately must produce its effects across a range of situations. The fact that the findings of Batterham et al. cannot easily be replicated calls into question the potential value of an anti-obesity approach that is based on administration of PYY3-36.

A reduced model of the fluorescence from the cyanobacterial photosynthetic apparatus designed for the in situ detection of cyanobacteria.

Fluorometric determination of the chlorophyll (Chl) content of cyanobacteria is impeded by the unique structure of their photosynthetic apparatus, i.e., the phycobilisomes (PBSs) in the light-harvesting antennae. The problems are caused by the variations in the ratio of the pigment PC to Chl a resulting from adaptation to varying environmental conditions. In order to include cyanobacteria in fluorometric analysis of algae, a simplified energy distribution model describing energy pathways in the cyanobacterial photosynthetic apparatus was conceptualized. Two sets of mathematical equations were derived from this model and tested. Fluorescence of cyanobacteria was measured with a new fluorometer at seven excitation wavelength ranges and at three detection channels (650, 685 and 720 nm) in vivo. By employing a new fit procedure, we were able to correct for variations in the cyanobacterial fluorescence excitation spectra and to account for other phytoplankton signals. The effect of energy-state transitions on the PC fluorescence emission of PBSs was documented. The additional use of the PC fluorescence signal in combination with our recently developed mathematical approach for phytoplankton analysis based on Chl fluorescence spectroscopy allows a more detailed study of cyanobacteria and other phytoplankton in vivo and in situ.

Impaired sodium excretion, decreased glomerular filtration rate and elevated blood pressure in endothelin receptor type B deficient rats.

The renal endothelin (ET) system, particularly the ET type B receptor, has been implicated in the regulation of sodium excretion and glomerular filtration rate (GFR). We analyzed kidney morphology and function in a rat strain characterized by complete absence of a functional ETB receptor. Due to Hirschsprung's disease limiting lifetime in these rats, studies were performed in 23-day-old rats. Kidney size and morphology (glomerular and interstitial matrix content, glomerular size and cell density and intrarenal vascular morphology) were normal in ETB-deficient rats. There were also no evidence of altered kidney cell cycle regulation in these rats. GFR was significantly lower, by 72% (P<0.001), in homozygous ETB-deficient rats than in wild-type rats. Fractional sodium excretion was likewise markedly reduced by 84% in homozygous ETB-deficient rats (P<0.001 versus wild-type rats). Treatment with the specific epithelial sodium channel blocker amiloride led to a much higher increase in fractional sodium excretion in ETB-deficient rats (934.2+/-73% in ETB-deficient rats versus 297+/-20% in wild-type rats, expressed as percentage of corresponding placebo treated control; P<0.001). Mean arterial blood pressure was elevated by 7.9 mmHg in homozygous ETB-deficient rats (P<0.05 versus wild-type rats). Our study demonstrates that ETB-deficiency causes early onset kidney dysfunction characterized by a markedly reduced sodium excretion, decreased GFR, and slightly elevated blood pressure. The complete absence of the ETB receptor causes in the kidney--in contrast to the colon--a functional rather than a developmental, neural crest cell dependent disease, since kidney morphology was normal in ETB-deficient rats. The much higher increase in the fractional sodium excretion in ETB-deficient rats after pharmacological blockade of the epithelial sodium channel indicates that the decreased fractional sodium excretion in ETB-deficient rats is most probably due to a lack of the inhibitory property of the ETB receptor on the epithelial sodium channel activity.

Regulation of the renal endothelin system in the two-kidney, one clip renal hypertensive rat.

Renovascular hypertension in the two-kidney, one clip (2K1C) model is characterized by initially elevated angiotensin-II (A-II) plasma concentrations, caused by ischemia in the clipped kidney and shear stress in the nonclipped kidney. These features are known stimuli of the endothelin (ET) system. The aim of this study was to analyze whether the renal ET system is activated in 2K1C renal hypertension in the rat. Wistar Kyoto rats (9 weeks old) were randomly assigned to four groups. Groups 1 and 3 underwent renal artery clipping, groups 2 and 4 were sham-operated. Groups 1 and 2 were used for analysis at 10 days after clipping, groups 3 and 4 12 weeks after clipping. We measured immunoreactive ET-1 renal tissue concentration as well as the ET(A)- and ET(B)-receptor density and affinity in renal cortex and medulla. We detected an increased immunoreactive ET-1 tissue concentration compared to the sham-operated control in the renal cortex of the nonclipped kidney 10 days (25.6 +/- 12.0 pg/g vs 12.5 +/- 5.0, 1 pg/g; p < 0.05) and in the renal cortex of the clipped kidney 90 days after clipping (92.4 +/- 47 pg/g vs 22.9 +/- 21 pg/g; p < 0.05), An increased ET(A)-receptor density was revealed in the renal medulla of the clipped kidney 10 days (624 +/- 130 fmol/mg vs 276 +/- 68 fmol/mg; p < 0.05) and 90 days (859 +/- 131 fmol/mg vs 493 +/- 93 fmol/mg; p < 0.05) after clipping. There were no differences in ET(B)-receptor density or binding affinity of either ET(A)- or ET(B)-receptors. In the 2K1C rat model of renovascular hypertension the renal ET system is activated. This activation is time-dependent and also dependent on the specific pathophysiological condition (clipped vs nonclipped). Increased ET-1 tissue concentration and upregulation of ET(A)-receptor density might lead to a synergistic activation of the ET system.

Pulmonary fibrosis and chronic lung inflammation in ET-1 transgenic mice.

The pulmonary endothelin (ET) system has been implicated in the pathogenesis of chronic lung diseases such as pulmonary hypertension, asthma, chronic obstructive lung disease, idiopathic pulmonary fibrosis, and bronchiolitis obliterans. However, the etiologic role of ET-1 in these diseases has not yet been established. We recently demonstrated that ET-1 transgenic mice, generated using the human prepro-ET-1 expression cassette including the cis-acting transcriptional regulatory elements, had predominant transgene expression in lung, brain, and kidney. We used these mice in the present study to analyze the pathophysiologic consequences of long-term pulmonary overexpression of ET-1. We found that ET-1 overexpression in the lungs did not result in significant pulmonary hypertension, but did result in development of a progressive pulmonary fibrosis and recruitment of inflammatory cells (predominantly CD4-positive cells). Our study provides evidence that a long-term activated pulmonary ET system, without any other stimuli, produces chronic lymphocytic inflammation and lung fibrosis. This suggests that overexpression of ET-1 may be a central event in the pathogenesis of lung diseases associated with fibrosis and chronic inflammation, such as pulmonary fibrosis and bronchiolitis.

ETA receptor blockade induces fibrosis of the clipped kidney in two-kidney-one-clip renovascular hypertensive rats.

BACKGROUND: In two kidney-one clip renovascular hypertension (2K1C), blood flow is reduced in the clipped kidney leading to ischaemia. The non-clipped kidney is characterized by increased shear stress. Circulating Ang II is elevated. All these factors are stimuli of the paracrine renal endothelin system. Indeed, we demonstrated an activation of the renal endothelin system in the 2K1C rat model. METHODS: We analysed the effects of chronic treatment with the ETA receptor antagonist BQ-123 on blood pressure, heart rate, plasma renin activity, and on the progression of glomerulosclerosis, interstitial fibrosis and vascular remodeling in the clipped and non-clipped kidney. RESULTS: Long-term treatment with BQ-123 led to a fibrotic atrophy of the clipped kidney characterized by a significantly reduced weight of the clipped kidney compared to the clipped kidney of the placebo-treated group. Computer-aided image analysis revealed a markedly enhanced interstitial fibrosis of these clipped kidneys after long-term ETA blockade. The effects of ETA receptor antagonists on the non-clipped kidney were less pronounced. Neither blood pressure nor plasma renin activity were significantly altered by BQ-123 treatment. CONCLUSIONS: The present study indicates that long-term blockade of the activated endothelin system in the clipped kidney of rats with renovascular hypertension using an ETA receptor antagonist led to a fibrotic atrophy of the clipped kidney.

Renal endothelin system in polycystic kidney disease.

Polycystic kidney disease (PKD) is characterized by interstitial fibrosis and formation of renal cysts. Interestingly, interstitial fibrosis and renal cyst formation were also seen in human endothelin-1 (ET-1) transgenic mice. This study, therefore, analyzes the tissue distribution of ET-1, the tissue concentrations of ET-1, as well as the expression of ET receptor subtypes in the kidneys of a rat model of PKD: Han:SPRD rats. Six-week-old heterozygous (cy/+) and homozygous (cy/cy), as well as 6-mo-old heterozygous (cy/+) Han:SPRD rats and the corresponding age-matched Sprague Dawley littermates (SD) (+/+) were analyzed. Furthermore, the acute effects of the mixed (A/B) endothelin receptor antagonist bosentan on hemodynamic and renal function were investigated in 6-mo-old, conscious, chronically instrumented (cy/+) rats. The kidneys of affected rats showed significantly elevated tissue levels of ET-1 compared with age-matched controls (3.5 +/- 0.3-fold in young cy/cy rats, P < 0.01; 1.4 +/- 0.2-fold in young cy/+ rats, P < 0.01; 6.2 +/- 0.4-fold in old cy/+ rats, P < 0.001) due to a highly increased ET-1 synthesis within the epithelial cells of the cysts. Analyzing tissue sections from patients with typical autosomal dominant PKD demonstrated a high ET-1 expression within the epithelial cells of the cysts as well. Scatchard analysis revealed a markedly decreased ETA and ETB receptor density in all groups of affected rats. The acute blockade of both endothelin receptor subtypes using bosentan in 6-mo-old heterozygous PKD rats led to a significant decrease in mean arterial BP (MAP) (-19.7 +/- 2.1 mmHg, P < 0.05) and GFR (-41 +/- 5%, P < 0.005). Renal blood flow (RBF) was significantly increased (+2.1 +/- 0.5 ml/min, P < 0.05) after bosentan, whereas bosentan had no effect on MAP, GFR, and RBF in age-matched controls. These data show that the paracrine renal endothelin system is activated in PKD and participates in the regulation of MAP, GFR, RBF, and possibly contributes to renal cyst formation and fibrosis.

Apoptosis in kidneys of endothelin-1 transgenic mice.

Endothelin-1 (ET-1) transgenic mice are characterized by age-dependent development of renal cysts and renal fibrosis (interstitial fibrosis and glomerulosclerosis), leading to a progressive decrease in glomerular filtration rate. The mechanism causing the loss of functionally and morphologically normal renal tissue is unknown. An imbalance between cell proliferation and apoptosis in the kidneys of ET-1 transgenic mice might contribute to this process. We identified apoptotic cells in kidney sections by in situ end-labeling and by the typical nuclear chromatin morphology after propidium iodide (PI) staining. Cell proliferation was measured by estimating the proliferating cell nuclear antigen (PCNA)-positive cells. The numbers of apoptotic cells were significantly increased (p < 0.001) in the kidneys of 14-month-old ET-1 transgenic mice, whereas cell proliferation was not enhanced. Apoptotic cells were detected in the glomeruli, tubular cells, and renal interstitial cells in ET-1 transgenic mice. In conclusion, apoptotic loss of functional renal tissue appears to be associated with the progression of cyst formation and renal fibrosis. Therefore, an imbalance between cell proliferation and apoptosis could be an important cellular mechanism in ET-1 transgenic mice leading to end-stage kidney disease.

The paracrine endothelin system: pathophysiology and implications in clinical medicine.

Apart from the initially described vasoconstriction, endothelins have been shown to cause a variety of biological activities in non-vascular tissues. A rapidly growing body of data supports the concept of endothelin as a paracrine acting hormone. In this review, we will discuss the impact of this local endothelin system for various cardiovascular pathophysiological states, especially atherosclerotic vascular disease, restenosis, myocardial infarction, congestive heart failure, and arterial hypertension. In addition, the endothelin system is a modulator of renal function via its binding to abundant receptors in renal tissue and by the ability of renal endothelial and epithelial cells to synthesize and release endothelin. In the kidney, endothelin may function as a paracrine/autocrine factor in the regulation of renal blood flow, glomerular haemodynamics, and sodium and water homeostasis. The renal endothelin system is involved in kidney diseases such as impaired renal function in liver cirrhosis, cyclosporin toxicity, acute renal failure and renal glomerular and interstitial fibrosis. Therapeutic approaches with new orally active endothelin receptor antagonists are also discussed.

Endothelin-1 transgenic mice develop glomerulosclerosis, interstitial fibrosis, and renal cysts but not hypertension.

The human endothelin-1 (ET-1) gene under the control of its natural promoter was transferred into the germline of mice. The transgene was expressed predominantly in the brain, lung, and kidney. Transgene expression was associated with a pathological phenotype manifested by signs such as age-dependent development of renal cysts, interstitial fibrosis of the kidneys, and glomerulosclerosis leading to a progressive decrease in glomerular filtration rate. This pathology developed in spite of only slightly elevated plasma and tissue ET-1 concentrations. Blood pressure was not affected even after the development of an impaired glomerular filtration rate. Therefore, these transgenic lines provide a new blood pressure-independent animal model of ET-1-induced renal pathology leading to renal fibrosis and fatal kidney disease.

Characterization of the renal phenotype of transgenic rats expressing the human endothelin-2 gene.

We have previously established a transgenic rat model termed TGR(hET-2)37 overexpressing the human endothelin-2 (ET-2) gene with high renal transgene expression. This renal overexpression is of pathophysiological interest because a long-term activated paracrine renal endothelin system has been implicated in chronic renal failure due to progressive glomerular injury. Therefore, our aim in the present study was to analyze renal transgene expression in detail and address the question of whether transgene expression causes phenotypic and functional changes in the kidney. We used reverse transcription-polymerase chain reaction and in situ hybridization techniques for transgene expression analysis. Tissue ET-2 concentrations were measured with a specific radioimmunoassay. For histological evaluation of renal tissue, all samples were subjected to hematoxylin-eosin and periodic acid-Schiff staining. Renal tissue ET-2 concentrations were significantly increased in TGR(hET-2)37 rats. Using in situ hybridization, we found that the human ET-2 gene was almost exclusively expressed within the glomeruli. The glomerular transgene expression resulted in a significantly increased glomerular injury score and likewise in a significantly increased protein excretion, whereas glomerular filtration rate was not altered. Blood pressure was similar in TGR(hET-2)37 rats and age-matched controls, suggesting that the local changes in the kidney were correlated with paracrine endothelin actions. In conclusion, our study revealed that the major renal expression site of the human ET-2 transgene in TGR(hET-2)37 rats was within the glomeruli and caused the development of glomerulo-sclerosis with significantly increased protein excretion that is independent of blood pressure. We suggest that TGR(hET-2)37 rats are a new monogenetic animal model for study of the paracrine renal endothelin system and its involvement in renal pathophysiology.

Role of the paracrine liver endothelin system in the pathogenesis of CCl4-induced liver injury.

This study analyzed if the paracrine liver endothelin system participates in the pathogenesis of CCl4-induced hepatotoxicity. Wistar Kyoto rats were divided into four groups: a bosentan (mixed endothelin ETA and ETB receptor antagonist) treated group with CCl4 intoxication, a vehicle treated group with CCl4 intoxication, a nontreated control group and a bosentan treated control group. Hepatotoxicity was assessed by determination of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) followed by histopathological examinations. Tissue endothelin-1 concentrations and expression of endothelin receptor subtypes were analyzed. The tissue levels of endothelin-1 in the liver of rats with CCl4 intoxication were significantly higher than those in normal rats. Scatchard analysis revealed no differences in the density and binding constant of endothelin ETA and ETB receptor between rats with CCl4 intoxication and controls. Bosentan treatment of rats undergoing CCl4 inhalation resulted in a significant protection against elevation of ALT, AST, LDH and bilirubin. Histopathological examination of live sections for necrotic, swollen and lipid-laden cells revealed findings that were in agreement with the serum enzyme data. In conclusion, this study showed that the paracrine endothelin system is involved in the pathogenesis of CCl4-induced hepatotoxicity and that the blockade of the stimulated liver endothelin systems reduces CCl4-induced liver injury.

Protective effects of the mixed endothelin receptor antagonist bosentan in rats with CCL4-induced liver injury.

We determined whether the paracrine liver endothelin (ET) system participates in the pathogenesis of CCl4-induced hepatotoxicity. Wistar-Kyoto rats were divided into four groups: a bosentan-treated group with CCl4 intoxication, a vehicle-treated group with CCl4 intoxication, a nontreated control group, and a bosentan-treated control group. Hepatotoxicity was assessed by determination of serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH). Tissue ET-1 concentrations and expression of endothelin receptor subtypes were analyzed. The liver tissue levels of ET-1 in rats with CCl4 intoxication were significantly higher than in normal rats. Scatchard analysis revealed no differences in the density and binding constants of ETA and ETB receptors between rats with CCl4 intoxication and controls. Bosentan treatment of rats undergoing CCl4 inhalation resulted in significant protection against elevation of ALT, AST, LDH, and bilirubin. In conclusion, this study showed that the paracrine ET system in involved in the pathogenesis of CCl4-induced hepatotoxicity and that blockade of the stimulated liver endothelin system reduces CCl4-induced liver injury.