Permanent CNI treatment for prevention of renal allograft rejection in sensitized hosts can be replaced by regulatory T cells.

Recent data suggest that donor-specific memory T cells (T(mem)) are an independent risk factor for rejection and poor graft function in patients and a major challenge for immunosuppression minimizing strategies. Many tolerance induction protocols successfully proven in small animal models e.g. costimulatory blockade, T cell depletion failed in patients. Consequently, there is a need for more predictive transplant models to evaluate novel promising strategies, such as adoptive transfer of regulatory T cells (Treg). We established a clinically more relevant, life-supporting rat kidney transplant model using a high responder (DA to LEW) recipients that received donor-specific CD4(+)/ 8(+) GFP(+) T(mem) before transplantation to achieve similar pre-transplant frequencies of donor-specific T(mem) as seen in many patients. T cell depletion alone induced long-term graft survival in naïve recipients but could not prevent acute rejection in T(mem)(+) rats, like in patients. Only if T cell depletion was combined with permanent CNI-treatment, the intragraft inflammation, and acute/chronic allograft rejection could be controlled long-term. Remarkably, combining 10 days CNI treatment and adoptive transfer of Tregs (day 3) but not Treg alone also induced long-term graft survival and an intragraft tolerance profile (e.g. high TOAG-1) in T(mem)(+) rats. Our model allows evaluation of novel therapies under clinically relevant conditions.

ATP6AP2 over-expression causes morphological alterations in the hippocampus and in hippocampus-related behaviour.

The (pro)renin receptor [(P)RR], also known as ATP6AP2 [ATPase 6 accessory protein 2], is highly expressed in the brain. ATP6AP2 plays a role in early brain development, adult hippocampal neurogenesis and in cognitive functions. Lack of ATP6AP2 has deleterious effects, and mutations of ATP6AP2 in humans are associated with, e.g. X-linked intellectual disability. However, little is known about the effects of over-expression of ATP6AP2 in the adult brain. We hypothesized that mice over-expressing ATP6AP2 in the brain might exhibit altered neuroanatomical features and behavioural responses. To this end, we investigated heterozygous transgenic female mice and confirmed increased levels of ATP6AP2 in the brain. Our data show that over-expression of ATP6AP2 does not affect adult hippocampal neurogenesis, exercise-induced cell proliferation, or dendritic spine densities in the hippocampus. Only a reduced ventricular volume on the gross morphological level was found. However, ATP6AP2 over-expressing mice displayed altered exploratory behaviour with respect to the hole-board and novel object recognition tests. Moreover, primary adult hippocampal neural stem cells over-expressing ATP6AP2 exhibit a faster cell cycle progression and increased cell proliferation. Together, in contrast to the known deleterious effects of ATP6AP2 depletion, a moderate over-expression results in moderate behavioural changes and affects cell proliferation rate in vitro.

Features of the metabolic syndrome in the spontaneously hypertriglyceridemic Wistar Ottawa Karlsburg W (RT1u Haplotype) rat.

The aim of this study was to characterize the Wistar Ottawa Karlsburg W ([WOKW] RT1u haplotype) rat in a cross-sectional study (up to 14 weeks of age) for traits with pathophysiological relevance to the metabolic syndrome in comparison to the Dark Agouti (DA) rat, to determine the age at which the WOKW rat begins to manifest the characteristics of the metabolic syndrome. The findings indicate that the WOKW rat is dyslipidemic (high serum triglycerides and low high-density lipoprotein [HDL] cholesterol), hyperinsulinemic, and obese. The interval between 8 and 10 weeks appears to be the crucial age after which the most dramatic changes were observed in the measured phenotypic traits in the WOKW rat, as well as the most expressive differences between the WOKW and DA strains. Considering the phenotypic differences between WOKW and DA rats, the DA rat provides an appropriate control strain for crossing studies with the WOKW rat, which might contribute to the explanation of the genetic basis for traits of the metabolic syndrome in this model.

Congenic BB.SHR rat provides evidence for effects of a chromosome 4 segment (D4Mit6-Npy approximately 1 cm) on total serum and lipoprotein lipid concentration and composition after feeding a high-fat, high-cholesterol diet.

Congenic BB.SHR (previously referred to as BB.LL) rats were generated by transferring the segment of chromosome 4 flanked by the D4Mit6 and Spr loci from the spontaneously hypertensive rat (SHR/Mol) onto the genetic background of the diabetes-prone BB/OK rat. In this study, the influence of the above-mentioned region of chromosome 4 on triglyceride, cholesterol, and phospholipid phenotypes after a high-fat, high-cholesterol diet was examined by comparison of BB.SHR congenic rats with BB/OK rats. BB/OK and BB.SHR had comparable concentrations of basal and postdietary serum insulin, as well as of basal total serum triglycerides and had an identical body weight and food intake at the beginning of the test period. However, after 4 weeks on the test diet, BB.SHR rats were significantly heavier than BB/OK rats and had significantly higher food intake and lower total serum triglyceride concentrations. The basal serum leptin level was significantly lower, but postdietary serum leptin concentration did not show a significant difference between the 2 strains. Furthermore, significantly higher basal total serum cholesterol and phospholipid levels were observed in BB.SHR rats, but this difference disappeared after feeding the high-fat, high-cholesterol diet. Postdietary high-density lipoprotein (HDL)(2) cholesterol and phospholipid levels were significantly elevated in BB.SHR rats when compared with BB/OK rats. The 2 strains also differed slightly, but significantly, with respect to the other HDL phospholipid concentrations. In addition to previously described differences between BB/OK and BB.SHR rats, the results of this study clearly show the impact of genes, lying within the transferred segment, on serum lipid phenotypes after high-fat, high-cholesterol diet.

Quantitative trait loci for blood glucose confirm diabetes predisposing and protective genes, Iddm4 and Iddm5r, in the spontaneously diabetic BB/OK rat.

Several crossing studies using diabetic BB/OK and diabetes-resistant rat strains have clearly shown that the MHC class-II-genes of the RT1u haplotype (Iddm1) and the lymphopenia (Iddm2) are essential but not sufficient for type 1 diabetes development. The search for additional diabetogenic genes revealed predisposing non-MHC genes, Iddm3 and Iddm4, and a diabetes protective gene, Iddm5r, cosegregating with diabetes in the BB/OK rat subline. These findings were based on cosegregation studies comparing allele frequencies between diabetic and non-diabetic cross hybrids. Since, type 1 diabetes is characterised by hyperglycaemia we analysed 22 diabetic and 43 non-diabetic [(BB x SHR)FI x BB] backcross hybrids (28M:37F) which were already homozygous for Iddml and Iddm2 to search for quantitative trait loci (QTLs) affecting blood glucose in BB/OK rats. The QTL analysis using 117 microsatellite markers located on 19 autosomal chromosomes and the X chromosome, revealed suggestive linkage for blood glucose at the same position for diabetics (lod score 3.1) and non-diabetics at an age of 16 weeks at locus D6Mgh2 on chromosome 6 (lod score 1.9). In contrast, the peak for nondiabetics at an age of 28 weeks (lod score 3.1) was located in the region on chromosome 1 flanked by D1Mgh12 and D1Mit14, whereas the peak for diabetics (lod score 1.9) was found between Sa and Igf2. The distance between two peaks is ca. 50 cM. These findings are consistent with previously described results and provide strong evidence on the relevance of the described region for the development of diabetes not only in the rat, but, regarding the chromosomal homology also in human.

Blood pressure, heart rate and motor activity in 6 inbred rat strains and wild rats (Rattus norvegicus): a comparative study.

Inbreeding for many generations under optimal environmental conditions may have favoured the survival of alleles for blood pressure increase in phenotypically normotensive rat strains. To prove this hypothesis we measured telemetrically systolic (SBP) and diastolic blood pressure (DBP), heart rate (HR) and motor activity (MA) in 6 inbred rat strains (BB, BN, LEW, DA, F344, WKY) and wild rats most probably possessing all of the alleles for normotension. For the first time it is shown that systolic blood pressure can significantly differ between normotensive inbred rat strains and that most probably some inbred rat strains will be characterised by a systolic blood pressure found in their progenitors, the wild rats. In addition, the typical night activity of rodents was not seen in 2 inbred rat strains. All findings together may be interpreted in the sense that most, if not all inbred rats strains have more or less disturbances in blood pressure, HR and/or MA and that there is most probably no "healthy" inbred rat strain available so that wild rats may be an alternative for crossing studies dissecting hypertension in particular and diseases in general.

Metabolic variability among disease-resistant inbred rat strains and in comparison with wild rats (Rattus norvegicus).

1. Inbreeding and optimization of environmental conditions for laboratory rats may have led to the survival of mutants with metabolic aberrations but without evident disease phenotype. Therefore, in the present study, we compared metabolic traits between so-called disease-resistant inbred rat strains Dark Agouti (DA), Brown Norway (BN), Lewis (LEW), Wistar-Kyoto (WKY), Fischer 344 (F344) and wild rats (Rattus norvegicus). 2. Twelve males of each strain at 12, 13 and 14 weeks of age were studied for bodyweight, body mass index (BMI), blood glucose, serum triglycerides, total cholesterol, insulin and leptin. 3. In comparison with wild rats, the cholesterol values were significantly increased in all inbred rats studied. Except for DA rats, all rats were also significantly heavier than wild rats. 4. There were also significant differences between the different disease-resistant strains and WKY rats were the biggest animals with the highest bodyweight, BMI and cholesterol values. 5. The strains could be separated into groups with either very high (F344, LEW, WKY) or low values (DA, BN) of serum insulin and leptin levels. 6. Because all rats were studied under the same conditions, the findings suggest a substantial strain dependence in feeding behaviour and energy balance caused by the different genotypes.

Metabolic syndrome and aging in Wistar Ottawa Karlsburg W rats.

BACKGROUND AND OBJECTIVE: Comparative studies have shown that Wistar Ottawa Karlsburg W (RT1u) rats (WOKW) develop a nearly complete metabolic syndrome with obesity, moderate hypertension, dyslipidemia, hyperinsulinemia, and impaired glucose tolerance up to an age of 28 weeks. Because metabolic data thereafter are missing, WOKW and disease-resistant DA rats were studied for 12 months beginning at an age of 5 months. METHODS: Eighteen male inbred WOKW and DA rats were studied monthly from the 5th to the 17th month of life for traits of the metabolic syndrome such as body weight, body mass index (BMI), serum triglycerides, total cholesterol, leptin, insulin as well as glucose tolerance, 24 h excretion of urine total protein and creatinine including telemetric measurement of blood pressure in six males per each group. RESULTS: Except for serum total cholesterol, the measured values for most traits studied were significantly higher in WOKW than in DA rats at an age of 5 months. At an age of 17 months all traits were significantly elevated in WOKW compared with DA rats. WOKW rats were hypertensive, dyslipidemic, obese, glucose intolerant, hyperinsulinemic and proteinuric. CONCLUSION: Considering the phenotype of the WOKW rat described until now and the fact that the metabolic syndrome in this rat is polygenetically determined, the WOKW rat is the most suitable animal model to study the pathophysiology of the facets of the syndrome.

Effects of quantitative trait loci for lipid phenotypes in the rat are influenced by age.

1. Previous study on the backcross hybrids derived from a cross of the spontaneously hypertensive rat (SHR/Mol) and the spontaneously diabetic BB/OK rat demonstrated the existence of quantitative trait loci (QTL) affecting lipid phenotypes on chromosome 4 and suggestive linkage of lipid phenotypes with markers on chromosome 1. Because the previous study was performed with backcross hybrids at 12 weeks of age and it is known that lipid phenotypes can show age-related differences, in the present study, the effect of QTL (chromosome 1 and 4) on serum triglycerides and cholesterol was longitudinally analysed between 20 and 32 weeks of age in backcross hybrids. 2. The results of the present study show that the effect of QTL on chromosome 4 (between It-6 and D4Mit9) for serum triglycerides was maximal at 20 weeks of age, but disappeared at 32 weeks of age. In contrast, the effect of QTL on serum total cholesterol on chromosome 4 (Npy-Spr) was maximal at 32 weeks of age. In contrast with the first study (12 weeks), the longitudinal investigation showed significant linkage of DIMit14 marker with lipid phenotypes on chromosome 1. 3. The results of the present study indicate the necessity of considering the role of age in QTL analysis of lipid phenotypes.

Genetic dissection of the syndrome X in the rat.

In 1988, Reaven used the term syndrome X to describe a relation between several disorders including hypertension, dyslipidemia, impaired glucose tolerance, obesity, and coronary heart disease. Despite a number of studies dealing with syndrome X, its genetic basis remains poorly understood. Regarding the complexity of this syndrome, it is important to use animal models developing the traits of the disease. Here we show a genetic dissection of syndrome X in the WOKW rat, an animal model of genetically determined syndrome X. We found a major quantitative trait locus (QTL) for glucose metabolism on chromosome 3 and further QTLs influencing obesity and body weight on chromosomes 1 and 5. Genetic determinants of dyslipidemia were mapped to chromosomes 4 and 17. In addition, suggestive linkage for serum insulin was found on chromosome 1 to the region previously shown to be associated with type-1 diabetes mellitus. This is the first study demonstrating independent genetic factors influencing traits of the syndrome X in the rat as well as a possible genetic relationships between syndrome X and diabetes mellitus. Moreover, regarding the close similarities between WOKW rat and human syndrome X, the study could help in a search of genetic factors involved in this complex metabolic disorder in human.

Metabolic features in disease-resistant as well as in spontaneously hypertensive rats and newly established obese Wistar Ottawa Karlsburg inbred rats.

OBJECTIVE: Studies, comparing several disease-prone and disease-resistant rat strains to elucidate the extent and severity of syndromes resembling human diseases are lacking. Therefore we studied the inbred rat strains BB/OK, BN/Crl, LEW/K and WKY/Crl in comparison with SHR/Mol and WOKW/K rats as models of metabolic syndrome. DESIGN: Body weight and body mass index (BMI) were measured in 12 males of each strain at 14 weeks. In addition blood glucose, serum triglycerides, cholesterol, insulin and leptin were determined at 12, 13 and 14 weeks of age. RESULTS: In contrast to SHR animals, WOKW rats develop a severe metabolic syndrome including obesity, hyperleptinemia, hyperinsulinemia and dyslipidemia. CONCLUSION: We conclude that; (i) the choice of disease-resistant inbred rat strains as 'healthy controls' for a disease-prone strain has to be carefully evaluated; (ii) in comparison with SHR, WOKW rats develop most if not all facets of the metabolic syndrome described in human and (iii) as with the human disease the syndrome in rats is polygenic.

Sex-specific and sex-independent quantitative trait loci for facets of the metabolic syndrome in WOKW rats.

WOKW rats develop a complete metabolic syndrome closely resembling human disease. Since genetic studies using male (WOKW x DA)F2 progeny showed that several independent genetic factors were involved, a polygenic basis for the syndrome in WOKW was assumed. However, because the metabolic syndrome in human clearly demonstrates sex differences, we have extended our study to include both male and female (WOKW x DA)F2 progeny in a genome-wide scan. Male- or female-specific quantitative trait loci (QTLs) were mapped for body weight, body mass index, adiposity index and serum insulin on chromosomes 1 and 5, serum triglycerides on chromosomes 4, 7, 11, and 16, serum total and high density lipoprotein cholesterol on chromosomes 3, 4, 5, 10, and 17, and serum leptin on chromosomes 8 and 16 as well as blood glucose and glucose tolerance (AUC) on chromosomes 3, 4 and 17. QTLs for both, males and females were only found for body weight on chromosome 1 and for serum total cholesterol on chromosome 3 and 10. These findings clearly demonstrate that there are sex-specific and sex-independent QTLs for facets of the metabolic syndrome in WOKW rats.

Quantitative trait loci for body weight, blood pressure, blood glucose, and serum lipids: linkage analysis with wild rats (Rattus norvegicus).

To study polygenetically inherited human diseases like hypertension, inbred rat strains are usually the preferred models. Because many inbred generations under optimized environmental conditions may have led to the survival of "silent" disease genes, we used a cross between one wild rat and genetically hypertensive SHR rats to analyze quantitative trait loci (QTLs) of blood pressure and related traits. The (Wild x SHR)F1 hybrids were transferred into a pathogen-free environment by wet-hysterectomy and were backcrossed onto SHR to generate first backcross hybrids (BC1). Progeny from one F1 female (n = 72) were phenotypically and genetically characterized to map QTLs. Significant, subsignificant, and suggestive evidence was found for more sex-specific than common linkage of blood pressure and most blood-pressure-related traits. Male- and female-specific regions were determined on different chromosomes for blood pressures (Chrs. 2 and 7 vs 5 and 11), body weight (Chrs. 10 vs 18), and blood glucose (Chr. 17 vs 20). A linkage in both males and females was shown for serum triglycerides on chromosomes 6 and 17, respectively, and blood glucose on chromosome 15. For serum total cholesterol, a significant linkage was found on chromosome 14 only in males. Our findings not only indicate the complex character of quantitative traits per se but also show impressively their dependence on sex, age, and strains in cosegregation analysis.

Genes of SHR rats protect spontaneously diabetic BB/OK rats from diabetes: lessons from congenic BB.SHR rat strains.

Diabetes in BB rats share many common features with human type 1 diabetes. One of them is the complex and polygenic nature of disease. Analysis of cross hybrids of diabetic BB/OK rats and rats of different diabetes-resistant strains has demonstrated that beside the MHC genes, Iddm1 and the lymphopenia, Iddm2, additional non-MHC genes are involved in diabetes development. To study the importance of the non-MHC genes, Iddm4 and Iddm3, two congenic BB.SHR rat strains were generated by recombining a segment of the SHR chromosome 6 (Iddm4; termed BB.6S; 15cM) or chromosome 18 (Iddm3; termed BB.18S; 24cM) into the BB/OK background by serial backcrossing and marker-aided selection. The characterization of both congenic strains demonstrates a drastic reduction of diabetes frequency in comparison to the BB/OK strain (86% vs 14% and 34%). It is supposed that diabetes protective genes of SHR must be located on both chromosomal segments and that these suppress the action of the essential and most important genes of diabetes development in the BB/OK rat, Iddm1, and Iddm2.

Congenic diabetes-prone BB.Sa and BB.Xs rats differ from their progenitor strain BB/OK in frequency and severity of insulin-dependent diabetes mellitus.

Two newly established congenic diabetes-prone BB rat strains designated BB.Sa and BB.Xs carrying a region of chromosome 1 (Sa-Lsn-Secr-Igf2-Tnt, 16 cM) and a region of chromosome X (DXMgh3-Mycs/Pfkb1-Ar, 36 cM) of the SHR rats, respectively, were studied to determine whether the transferred chromosomal regions influence diabetes frequency, age at onset, and clinical picture. Therefore, 4 complete litters of BB/OK (n = 43), BB.Sa (n = 45), and BB.Xs (n = 41) were observed for diabetes occurrence up to the age of 30 weeks. From these litters 6 diabetic males of each strain manifesting in an interval of 1 week were chosen to study body weight, blood glucose, insulin requirement to survive, and several diabetes-related serum constituents at onset of diabetes and after a diabetes duration of 150 days. The diabetes frequency was significantly lower in BB.Xs than in rats of the parental strain BB/OK, whereas comparable frequencies were found between BB/OK and BB.Sa rats. Obvious differences were observed 150 days after diabetes onset between BB/OK and both BB.Sa and BB.Xs rats. BB/OK rats were significantly heavier and needed significantly more insulin/100 g body weight than BB.Sa and BB.Xs rats. Comparisons of the serum constituents as lipids, proteins, and minerals revealed significant differences between diabetic BB/OK rats and their diabetic congenic derivatives in several traits studied at onset and after 150 days of insulin treatment. These results not only show the power of congenic lines in diabetes research, but indicate for the first time that there are genetic factors on chromosomes 1 and X influencing frequency and severity of diabetes in the BB/OK rat.

Phenotypic selection: a successful strategy to fix major genes of hypertension.

Hypertension is dominantly inherited in cross hybrids between hypertensive SHR/Mol and normotensive BB/OK rats. We used these cross hybrids for repeated backcrossing of selected hypertensive animals onto normotensive BB/OK rats to fix high blood pressure and to generate a hypertensive and diabetic BB/OK rat subline. After 8 backcrosses, the backcross parents were genetically analysed with the aid of 259 microsatellite markers to identify SHR genes causing blood pressure of 177 +/- 10 mmHg in this BB/OK rat subline. Loci on chromosomes 1, 14 and 18 showed longest heterozygosity. These loci might contain major genes of the SHR rat causing hypertension in this BB/OK rat subline. This classical strategy seems to be most suitable to fix major genes of hypertension in particular and complex traits in general and therefore to generate new animal models.