We investigated whether interleukin-1 (IL-1) activity in the rat hypothalamus was increased by immobilization stress (IS), and whether pretreatment with an interleukin-1 receptor antagonist (IL-1Ra) is capable of inhibiting IS-induced elevations of hypothalamic norepinephrine (NE), dopamine (DA), and serotonin (5-HT) and the levels of their metabolites as well as of plasma adrenocorticotropic hormone (ACTH). IL-1 activity was estimated with a bioassay using mouse thymocyte proliferation in the presence of concanavalin A. IL-1Ra was administered directly into the anterior hypothalamus, and monoamines were determined using a microdialysis technique and an HPLC system. First, we found that levels of IL-1 activity in the rat hypothalamus reached a maximum at 60 min after starting IS. Second, IL-1Ra (2 micrograms) significantly inhibited IS-induced increases in hypothalamic NE, DA, and 5-HT levels as well as the levels of their metabolites. In addition, IL-1Ra (2 micrograms) also inhibited the IS- induced elevation of plasma ACTH levels. Third, timing effects of IL- 1Ra administration on the IS-induced monoamines or ACTH responses were examined. IL-1Ra (2 micrograms) administered at 5 or 60 min before the start of IS, but not at 5 or 60 min after IS had been started, exerted inhibitory effects on these responses, indicating that the effects of IL-1 occurred within 5 min after the initiation of IS. In summary, these results suggest that IS enhances biologically active IL-1 in the hypothalamus, and that hypothalamic IL-1 plays a role in the regulation of IS-induced responses including elevated monoamine release in the hypothalamus and activation of the hypothalamo-pituitary-adrenal axis. Moreover, since 5 min is too short a time for IS to induce production of IL-1, IS may augment the effects of preexisting IL-1 in the hypothalamus.

Pharmacogenomics offers the potential to define metabolic pathways and to provide increased knowledge of drug actions. We studied relative levels of gene expression in the rat using a microarray with 8448 rat UniGenes (1928 known genes, 6520 unknown ESTs) in the liver and kidney as a function of time of day and then of feeding regime, which are common variables in preclinical pharmacogenomic studies. We identified 597 genes, including several key metabolic pathways, whose relative expression levels are significantly affected by time of day: expression of some was further modified by feeding state. These would have sparked interest in a pharmacogenomic study. Our study demonstrates that two common variables in pharmacogenomic studies can have dramatic effects on gene expression. This study provides investigators with baseline information for both kidney and liver with respect to 'normal' changes in gene expression influenced by time of day and feeding state. It also identifies 18 new genes that should be investigated for a role in circadian rhythms in peripheral tissues.

The lack of an appropriate animal model that spontaneously develops diabetic nephropathy has been a significant limitation in the search for genetic factors underlying this disease and the development of new therapeutic strategies to prevent progressive renal disease in diabetes. We introgressed the mitochondria and some passenger loci from the FHH/EurMcwi rat into the genetic background of diabetic GK rats, creating a new rat strain, T2DN (T2DN/Mcwi). Despite the high degree of genetic similarity between T2DN and GK rats (97% at 681 loci), diabetes ensues earlier and progresses more severely in T2DN rats. T2DN rats exhibit proteinuria by 6 months of age, accompanied by renal histologic abnormalities such as focal glomerulosclerosis, mesangial matrix expansion, and thickening of basement membranes. These characteristics progress over time, and nearly all T2DN rats exhibit diffuse global glomerulosclerosis with nodule formation and arteriolar hyalinosis by 18 months of age. The histologic changes in the kidney of T2DN rats closely mimic the changes seen in the kidney of patients with diabetes. These results indicate that the T2DN rat is a suitable model for investigating diabetic nephropathy. Here we report the initial genetic and physiological characterization of this new rat model of diabetic nephropathy. Diabetes 53: [735][736][737][738][739][740][741][742] 2004 O ne of the major morbidity and mortality factors confronted by patients with diabetes is an increased risk of developing diabetic nephropathy that often progresses to end-stage renal disease (ESRD) (1-3). A long-standing question pertaining to the development of renal disease in diabetes concerns the mechanisms involved in this process. A wealth of data have been generated on possible mechanisms by which diabetes and its ancillary metabolic, hemodynamic, growth, and glomerular cell injury-related alterations may modulate the progression of diabetic nephropathy (4 -7). Nevertheless, the observation that approximately two-thirds of patients with diabetes do not develop renal disease indicates that hyperglycemia is a permissive factor in diabetic nephropathy, but elevated plasma glucose levels alone do not fully account for renal injury (1). Thus, genetic factors are thought to play an important role in the susceptibility for diabetic nephropathy, with several clinical and epidemiologic studies supporting this hypothesis (8 -10).The complex interplay between diabetes-dependent and -independent factors in determining the progression of renal disease could become more amenable to study if there were adequate animal models that spontaneously develop diabetes and renal lesions that mimic those seen in patients with diabetic nephropathy. However, to date, no rodent model of diabetes that fully recapitulates the chronology of events and histologic changes in the kidney that are characteristic of patients with diabetic nephropathy has been developed. Several rodent models of spontaneous diabetes, such as the Zucker rat, BB rat, and DB mice, exhibit glomerula...

An in vitro culture of human fetal hepatocytes has been employed for infection by hepatitis B virus (HBV) virions that are produced by an established human hepatoma cell line, HB 611. HBV surface antigen and e antigen were released into the medium 3-4 days after infection, and production continued thereafter. RNA synthesis with similar kinetics was observed. Viral DNA replication started 2 days after infection, and replicative HBV DNA that included relaxed circles, single-stranded minus strands, and closed circles accumulated during 16 days of incubation. Immunofluorescent study using fluorescein isothiocyanate-labeled rabbit antisera directed against HBV core antigen revealed that this antigen is present in the nuclei in 12% of the infected cells. Particles containing HBV DNA were detected in the culture medium and were infectious. Thus, this in vitro infection system closely mimics infection in vivo and it allows detailed studies on early events associated with human HBV entry into cells and subsequent replication and integration.Hepatitis B virus (HBV) is a world-wide pathogen that causes hepatitis. Infection with the virus may be chronic and is associated with subsequent development of hepatocellular carcinoma. Although chimpanzees can be infected in vivo with HBV (1) and woodchucks, ground squirrels, and Peking ducks can be infected with their respective HBV-like viruses (2-4), use of whole animals poses certain limitations for detailed molecular studies. The most vexing technical problem in the biology of HBV has been lack of a practical in vitro system that allows infection and replication of this virus under controlled experimental conditions. Several investigators have attempted to infect human hepatic cells by using human adult and fetal hepatocytes (5, 6), fetal hepatocyte organ culture (7), oval cells originating from human liver (8), and HeLa cells (6). However, no signs of viral penetration, replication, or particle production have been observed except for a transient expression of some viral markers.We have established a cell line (HB 611) derived from a human hepatocellular carcinoma cell line (Huh6c15) by transfecting with a plasmid containing tandemly arranged HBV DNA. The HB 611 cells carry chromosomally integrated HBV genomes, and they allow expression of viral RNA, DNA, and proteins and produce Dane-particle-like virions (9). These particles have all the properties characteristic of the Dane particles from the patient's blood (10 MATERIALS AND METHODS Virus Sources and Cell Culture. The cell line HB 611, which produces Dane-particle-like double-shelled particles from integrated HBV DNA, has been described (9). A monolayer cell culture of HB 611 in 10% fetal calf serum/Dulbecco's modified Eagle's medium (10% FCS/DMEM) kept at 37°C was used as the source of infection.Hepatocytes were isolated from human fetal liver by a modification of the methods of Leffert and Paul (13). The liver tissuie was placed immediately in cold 10% FCS/DMEM supplemented with penicillin (100 units/ml) and s...

The genetically hypertensive fawn-hooded (FHH/Eur) rat is characterized by the early presence of systolic and glomerular hypertension, progressive proteinuria (UPV), and albuminuria (UAV), and focal glomerulosclerosis, resulting in premature death from renal failure. Previous studies showed that at least five genetic loci (Rf-1 to Rf-5) were linked to the development of renal impairment. Of these five, Rf-1 appears to play a major role. To study the impact of Rf-1 in the absence of the other loci, we transferred the Rf-1 region of chromosome 1, between the markers D1Mit34 and D1Rat156, Rf-1B for short, onto the genomic background of the normotensive August x Copenhagen Irish (ACI) rat. In this congenic strain, named ACI.FHH-D1Mit34/Rat156 or ACI.FHH-Rf1B, we challenged the renal hemodynamic function of these animals by studying the effects of unilateral nephrectomy (UNX) alone, or combined with N(G)-nitro-L-arginine methyl ester (L-NAME)-induced hypertension. Following UNX, the congenic strain developed significantly more UPV and UAV than the ACI progenitor. The differences were even more pronounced when UNX was combined with an L-NAME-induced rise in systolic blood pressure to about 150 mmHg, i.e., the level of hypertension present in the parental FHH strain. These findings indicate that the Rf-1B region of the FHH rat contains at least one gene affecting the susceptibility to progressive renal failure, especially in the presence of an increase in blood pressure.

Animal models have been used primarily as surrogates for humans, having similar disease-based phenotypes. Genomic organization also tends to be conserved between species, leading to the generation of comparative genome maps. The emergence of radiation hybrid (RH) maps, coupled with the large numbers of available Expressed Sequence Tags (ESTs), has revolutionized the way comparative maps can be built. We used publicly available rat, mouse, and human data to identify genes and ESTs with interspecies sequence identity (homology), identified their UniGene relationships, and incorporated their RH map positions to build integrated comparative maps with >2100 homologous UniGenes mapped in more than one species (approximately 6% of all mammalian genes). The generation of these maps is iterative and labor intensive; therefore, we developed a series of computer tools (not described here) based on our algorithm that identifies anchors between species and produces printable and on-line clickable comparative maps that link to a wide variety of useful tools and databases. The maps were constructed using sequence-based comparisons, thus creating "hooks" for further sequence-based annotation of human, mouse, and rat sequences. Currently, this map enables investigators to link the physiology of the rat with the genetics of the mouse and the clinical significance of the human.

To perform immunohistochemical study of the distribution of gamma-glutamyl transpeptidase in human organs, a highly specific antibody against the human enzyme is required. We prepared monoclonal antibody against gamma-glutamyl transpeptidase from human kidney, using the hybridoma technique. The antibody was of the IgG1 type and the light chain belonged to the kappa class. The antibody reacted specifically with the 63 KD heavy subunit of the enzyme. Examination of the specificity of the antibody performed by immunohistochemical staining of human kidney sections revealed that the antigen was localized on the brush border and along the basolateral membrane of the epithelial cells of both the convoluted and the straight portions of the proximal tubule. This antibody was also reactive in several human organs other than kidney, including epididymis, prostate, seminal vesicle, pancreas, and normal liver, and in human hepatoma. These findings indicate the existence of an antigenic determinant common to human kidney and other organs. The monoclonal antibody did not crossreact with mouse, rat, guinea pig, rabbit, or pig kidney.

No abstract