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Linkage analyses of crosses of rats susceptible to renal damage, fawn-hooded hypertensive (FHH), and those resistant to kidney damage, August x Copenhagen Irish (ACI), indicated that five quantitative trait loci (QTLs), Rf-1 to Rf-5, influence proteinuria (UPV), albuminuria (UAV) and focal glomerulosclerosis (FGS). Here we present data obtained in congenic rats to directly assess the role of the Rf-1 and Rf-5 QTLs.Renal damage (UPV, UAV, and FGS) was assessed in ACI, ACI.FHH-(D1Rat324-D1Rat156)(Rf-1B), and ACI.FHH-(D17Rat117-D17Arb5)(D17Rat180-D17Rat51) (Rf-5) congenic rats in the two-kidney (2K) control situation, and following L-NAME-induced hypertension, unilateral nephrectomy (UNX), and UNX combined with L-NAME. In addition we investigated renal blood flow (RBF) autoregulation in 2K congenic and parental ACI and FHH rats.Compared to ACI, Rf-1B congenic rats showed a significant increase in susceptibility to renal damage after all three treatments. The increase was most pronounced after UNX with L-NAME. In contrast, the degree of renal damage in Rf-5 congenic rats was not different from the ACI. Like FHH, Rf-1B rats had impaired renal autoregulation. In contrast, RBF autoregulation of Rf-5 rats does not differ from ACI.The Rf-5 QTL does not show any direct effect. The Rf-1 QTL carries one or more genes impairing renal autoregulation and influencing renal damage susceptibility. Whether these are the same genes remains to be established.

We report that Rab38 , a gene within the Rf-2 locus appears to influence the development of proteinuria (UPV) and albuminuria (UAV) in fawn-hooded hypertensive rats (FHH). Using congenic animals, we narrowed the region to eight genes; however, only one gene had a sequence variant. Rab38 has a

Hearts from Brown Norway (BN/Mcw) rats are more resistant to ischemia than hearts from Dahl S (SS/Mcw) rats. We determined whether nitric oxide (·NO) is responsible for increased cardioprotection in BN/Mcw vs. SS/Mcw hearts. Hearts from the two strains were treated with N G -monomethyl- l -arginine ( l -NMA) or S -nitrosoglutathione (GSNO) before ischemia and reperfusion. Infarct size in untreated BN/Mcw hearts was ~63% less than in SS/Mcw hearts. Inhibiting NOS with l -NMA increased infarct size in BN/Mcw hearts to that observed in untreated SS/Mcw hearts but did not further increase injury in SS/Mcw hearts. The ·NO donor GSNO decreased infarct size in SS/Mcw rats but had no effect on BN/Mcw hearts. Plasma and heart tissue from BN/Mcw rats contained 80% and 130% more nitrite + nitrate than that from SS/Mcw rats. These data suggest that increased ·NO production protects BN/Mcw hearts from ischemic injury. Real time PCR showed no differences in NOS1, NOS2 or NOS3 isozyme transcripts in the hearts from the two strains. NOS3 was the only isozyme detected by western analysis. Both strains exhibited the same level of NOS3 and hsp90 protein expression. However, hsp90 association with NOS3 in BN/Mcw hearts was increased twofold compared with SS/Mcw hearts. Inhibiting hsp90-NOS3 interaction with geldanamycin decreased the resistance to ischemia in BN/Mcw hearts but not in SS/Mcw hearts. SS/Mcw hearts also generated three times more N ω -nitro- l -arginine-methylester inhibitable superoxide than BN/Mcw hearts. These findings indicate that hsp90 with NOS3 increases ·NO production and decreases uncoupled NOS3 activity. We conclude increased association of hsp90 with NOS3 is a major mechanism by which BN/Mcw hearts are more resistant to ischemia than SS/Mcw hearts.

We aligned Incyte ESTs and publicly available sequences to the rat genome and analyzed rat chromosome 1q43-54, a region in which several quantitative trait loci (QTLs) have been identified, including renal disease, diabetes, hypertension, body weight, and encephalomyelitis. Within this region, which contains 255 Ensembl gene predictions, the aligned sequences clustered into 568 Incyte genes and gene fragments. Of the Incyte genes, 261 (46%) overlapped 184 (72%) of the Ensembl gene predictions, whereas 307 were unique to Incyte. The rat-to-human syntenic map displays rearrangement of this region on rat chr. 1 onto human chromosomes 9 and 10. The mapping of corresponding human disease phenotypes to either one of these chromosomes has allowed us to focus in on genes associated with disease phenotypes. As an example, we have used the syntenic information for the rat Rf-1 disease region and the orthologous human ESRD disease region to reduce the size of the original rat QTL to only 11.5 Mb. Using the syntenic information in combination with expression data from ESTs and microarrays, we have selected a set of 66 candidate disease genes for Rf-1. The combination of the results from these different analyses represents a powerful approach for narrowing the number of genes that could play a role in the development of complex diseases.

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.

The laboratory rat (Rattus norvegicus) is an indispensable tool in experimental medicine and drug development, having made inestimable contributions to human health. We report here the genome sequence of the Brown Norway (BN) rat strain. The sequence represents a high-quality 'draft' covering over 90% of the genome. The BN rat sequence is the third complete mammalian genome to be deciphered, and three-way comparisons with the human and mouse genomes resolve details of mammalian evolution. This first comprehensive analysis includes genes and proteins and their relation to human disease, repeated sequences, comparative genome-wide studies of mammalian orthologous chromosomal regions and rearrangement breakpoints, reconstruction of ancestral karyotypes and the events leading to existing species, rates of variation, and lineage-specific and lineage-independent evolutionary events such as expansion of gene families, orthology relations and protein evolution.

Resistance to thyroid hormone (RTH) is a syndrome in which patients have elevated thyroid hormone (TH) levels and decreased sensitivity to its action. We describe a child with extreme RTH and a severe phenotype. A 22-month-old female presented to the NIH with goiter, growth retardation, short stature, and deafness. Additionally, the patient had hypotonia, mental retardation, visual impairment, and a history of seizures. Brain magnetic resonance imaging showed evidence of demyelination and bilateral ventricular enlargement. The patient had markedly elevated free T3 and free T4 levels of more than 2000 pg/dl (normal, 230-420 pg/dl) and more than 64 pmol/liter (normal, 10.3-20.6 pmol/liter), respectively, and TSH of 6.88 mU/liter (normal, 0.6-6.3 mU/liter). These are the highest TH levels reported for a heterozygous RTH patient. A T3 stimulation test confirmed the diagnosis of RTH in the pituitary and peripheral tissues. Molecular analyses of the patient's genomic DNA by PCR identified a single base deletion in exon 10 of her TRbeta gene that resulted in a frameshift and early stop codon. This, in turn, encoded a truncated receptor that lacked the last 20 amino acids. Cotransfection studies showed that the mutant TR was transcriptionally inactive even in the presence of 10(-6) M T3 and had strong dominant negative activity over the wild-type receptor. It is likely that the severely defective TRbeta mutant contributed to the extreme RTH phenotype and resistance in our patient.

Anti-epiligrin cicatricial pemphigoid (AECP) is a mucosal-predominant, subepithelial blistering disease characterized by IgG anti-basement membrane autoantibodies to laminin 5 (alpha3beta3gamma2). This and prior studies found that autoantibodies from most patients recognize the alpha-subunit of this laminin isoform. Accordingly, sera from 10 representative patients were tested against prokaryotic recombinants of this polypeptide in epitope mapping studies. cDNAs spanning the full length of the alpha-subunit were generated by PCR, directionally cloned into the pGEX-4T-3 vector, and expressed as glutathione-S-transferase fusion proteins of appropriate size and immunoreactivity. Sera from 9 of 10 AECP patients immunoblotted fusion proteins corresponding to subdomains G2, G3, G4, and G5 at the carboxyl terminus of the laminin 5 alpha-subunit. Serum from 1 patient (and that from normal volunteers) showed no reactivity to any fusion proteins; no sera bound recombinant glutathione-S-transferase alone. Immunoadsorption of patient sera with fusion proteins corresponding to the G domain substantially reduced basement membrane autoantibody titers. IgG from patients with this form of cicatricial pemphigoid recognize the portion of laminin 5 thought to play a key role in promoting keratinocyte adhesion to epidermal basement membrane.

The U.S. Congress has passed legislation requiring the EPA to implement screening tests for identifying endocrine-disrupting chemicals. A series of workshops was sponsored by the EPA, the Chemical Manufacturers Association, and the World Wildlife Fund; one workshop focused on screens for chemicals that alter thyroid hormone function and homeostasis. Participants at this meeting identified and examined methods to detect alterations in thyroid hormone synthesis, transport, and catabolism. In addition, some methods to detect chemicals that bind to the thyroid hormone receptors acting as either agonists or antagonists were also identified. Screening methods used in mammals as well as other vertebrate classes were examined. There was a general consensus that all known chemicals which interfere with thyroid hormone function and homeostasis act by either inhibiting synthesis, altering serum transport proteins, or by increasing catabolism of thyroid hormones. There are no direct data to support the assertion that certain environmental chemicals bind and activate the thyroid hormone receptors; further research is indicated. In light of this, screening methods should reflect known mechanisms of action. Most methods examined, albeit useful for mechanistic studies, were thought to be too specific and therefore would not be applicable for broad-based screening. Determination of serum thyroid hormone concentrations following chemical exposure in rodents was thought to be a reasonable initial screen. Concurrent histologic evaluation of the thyroid would strengthen this screen. Similar methods in teleosts may be useful as screens, but would require indicators of tissue production of thyroid hormones. The use of tadpole metamorphosis as a screen may also be useful; however, this method requires validation and standardization prior to use as a broad-based screen.

Hearts from Brown Norway (BN) rats are more resistant to ischemia-reperfusion (IR) injury than hearts from Dahl Salt Sensitive (SS) rats. Moreover, introgression of BN chromosome 6 into SS confers IR resistance to the same degree as BN. We hypothesize that mitochondrial complex I preservation is linked to cardioprotection in this consomic rat model.Langendorff-prepared hearts from eight week old male BN, SS6BN and SS rats were subjected to 20 min perfusion, 30 min global ischemia or no ischemia (control), and 30 min reperfusion. Mitochondria were isolated and mitochondrial complex I preservation was measured by Western blotting using complex I NDUFA9 subunit antibody.We found higher protein levels in ischemic BN and SS6BN rat mitochondria compared to SS suggesting better preservation of complex I in BN and SS6BN. In contrast, the non-ischemic groups showed no detectable difference in protein levels.Previous studies have shown that mitochondrial function can be a trigger as well as an effector of cardioprotection. Our data shows that mitochondrial complex I is better preserved in BN and SS6BN than in SS. Since myocardial and mitochondrial functions differ significantly between the more IR-susceptible SS and the more IR-resistant BN and SS6BN strains, this suggests that rat chromosome 6 encodes one or more genes that are critical for mitochondrial function and cardioprotection.

By means of in vivo and in vitro approaches, we studied the effect of thyroid hormone on postnatal development of rat testis. T3 treatment in neonatal rats is associated with an increase of testis size of about 60%, compared to coeval controls. Increased number of both Sertoli and germ cells and enlarged diameter of seminiferous cords were found in hyperplastic testes. In the T3-treated group, TSH serum levels were low and a slight increase of FSH was found. In vitro treatment of neonatal testis fragments by 10(-7) M T3 for 3 days increased the number of gonocytes (P < 0.001 vs control) and decreased the percentage of degenerating germ cells (P < 0.001 vs control). In the adult testis, both in vivo and in vitro treatments with thyroid hormone did not induce morphological modifications, thus demonstrating that the critical window of thyroid hormone effectiveness coincides with the prepuberal period. Since thyroid hormone stimulates Sertoli cells to secrete growth factors and nutrients for germ cell development, we suggest that the increased testicular size and germ cell number following the T3 treatment is mediated by a direct hormonal effect on the somatic cell of the seminiferous epithelium.

Background Ghrelin is the endogenous ligand for the growth hormone secretagogue receptor (GHSR). Ghrelin regulates feeding activity and interdigestive contractions of the stomach in rodents. To investigate the role of endogenous ghrelin in the digestive system, we have developed GHSR-mutant rats, named FHH-Ghsrm1Mcwi, using the Fawn-Hooded Hypertensive (FHH) parental strain.

The FHH (fawn-hooded hypertensive) rat is a model of hypertension-associated chronic kidney damage. Five interacting quantitative trait loci (QTLs), named Rf-1 – Rf-5 , determine the high renal susceptibility. The aim of the present study was to investigate a possible interaction between Rf-1 and Rf-3 . Differences in renal susceptibility between ACI (August × Copenhagen Irish) controls, Rf-1A and Rf-3 single congenics, and Rf-1A + 3 double congenic rats were assessed using four different treatments: two-kidney control (2K), 2K plus N ω -nitro-L-arginine methyl ester (L-NAME)-induced hypertension (2K+L-NAME), unilateral nephrectomy (UNX), and UNX plus L-NAME-induced hypertension (UNX+L-NAME). Proteinuria (UPV) and systolic blood pressure (SBP) were assessed after 6, 12, and 18 weeks, while the incidence of glomerulosclerosis (%FGS) was determined at the end of the experiment. In a separate experiment, renal autoregulation was assessed in 13–15-week old 2K rats of all four strains. Compared to ACI rats, small increases in renal susceptibility were found in Rf-1A and Rf-3 single congenics following 2K+L-NAME, UNX, and UNX+L-NAME treatments. However, in the Rf-1A + 3 double congenics, a major increase in renal susceptibility was found with all four treatments. Both Rf-1A and Rf-1A + 3 congenic rats had an impaired renal autoregulation. In contrast, the Rf-3 had a normal autoregulation, similar to that of the ACI rat. These findings indicate that Rf-1 and Rf-3 alone slightly increase the susceptibility to the development of renal damage. However, a synergistic interaction between these two QTLs markedly enhances renal susceptibility. In contrast to the Rf-1 region, the Rf-3 region does not carry genes influencing renal autoregulation.

SPONASTRIME dysplasia is an autosomal-recessive spondyloepimetaphyseal dysplasia characterized by spine (spondylar) abnormalities, midface hypoplasia with a depressed nasal bridge, metaphyseal striations, and disproportionate short stature. Scoliosis, coxa vara, childhood cataracts, short dental roots, and hypogammaglobulinemia have also been reported in this disorder. Although an autosomal-recessive inheritance pattern has been hypothesized, pathogenic variants in a specific gene have not been discovered in individuals with SPONASTRIME dysplasia. Here, we identified bi-allelic variants in TONSL, which encodes the Tonsoku-like DNA repair protein, in nine subjects (from eight families) with SPONASTRIME dysplasia, and four subjects (from three families) with short stature of varied severity and spondylometaphyseal dysplasia with or without immunologic and hematologic abnormalities, but no definitive metaphyseal striations at diagnosis. The finding of early embryonic lethality in a Tonsl-/- murine model and the discovery of reduced length, spinal abnormalities, reduced numbers of neutrophils, and early lethality in a tonsl-/- zebrafish model both support the hypomorphic nature of the identified TONSL variants. Moreover, functional studies revealed increased amounts of spontaneous replication fork stalling and chromosomal aberrations, as well as fewer camptothecin (CPT)-induced RAD51 foci in subject-derived cell lines. Importantly, these cellular defects were rescued upon re-expression of wild-type (WT) TONSL; this rescue is consistent with the hypothesis that hypomorphic TONSL variants are pathogenic. Overall, our studies in humans, mice, zebrafish, and subject-derived cell lines confirm that pathogenic variants in TONSL impair DNA replication and homologous recombination-dependent repair processes, and they lead to a spectrum of skeletal dysplasia phenotypes with numerous extra-skeletal manifestations.

Interaction between Rf-1 and Rf-4 quantitative trait loci increases susceptibility to renal damage in double congenic rats. Background Five quantitative trait loci (QTLs), Rf-1 to Rf-5 , were found in Fawn-Hooded hypertensive (FHH) rats influencing susceptibility to renal damage. Previously, we found that single transfer of the Rf-1 QTL from FHH rats onto the renal-resistant August × Copenhagen Irish (ACI) strain caused a small increase in renal susceptibility. To investigate the separate role of the Rf-4 QTL and its interaction with Rf-1 , we generated a single congenic strain carrying Rf-4 and a double congenic carrying both Rf-1 and Rf-4 . Methods Differences in renal susceptibility between ACI, Rf-1A , and Rf-4 single congenics and Rf-1A+4 double congenics were assessed using four different treatments: control (two-kidney), two-kidney with L-arginine analogue N-nitro-L-arginine methyl ester (L-NAME)-induced hypertension, unilateral nephrectomy, and unilateral nephrectomy + L-NAME. In separate experiments, renal blood flow (RBF) autoregulation was compared between two-kidney ACI and congenic rats. Results Compared to ACI, Rf-1A rats developed more renal damage, while Rf-4 rats did not. The most severe renal damage was found in the Rf-1A+4 double congenic rats. Analysis of variance (ANOVA) demonstrated a significant interaction between the Rf-1A and Rf-4 QTLs. The magnitude of the interaction varied with the type and duration of the treatment. The RBF autoregulation was impaired in Rf-1A single and Rf-1A+4 double congenics, while in Rf-4 single congenics it was similar to that of ACI controls. Conclusion These findings indicate that the Rf-1 QTL directly influences renal susceptibility and autoregulation. In contrast, the Rf-4 QTL shows no direct effects, but significantly increases susceptibility to renal damage via an interaction with Rf-1 .

Backgound The male-specific region of chromosome-Y (MSY) contributes to phenotypes outside of testis development and has a high rate of evolution between mammalian species. With a lack of genomic crossover, MSY is one of the few genomic areas under similar variation and evolutionary selection in inbred and outbred animal populations, allowing for an assessment of evolutionary mechanisms to translate between the populations. Methods Using next-generation sequencing, MSY consomic strains, molecular characterization, and large-scale phenotyping, we present here regions of MSY that contribute to inbred strain phenotypes. Results We have shown that (1) MSY of rat has nine autosomal gene transposition events with strain-specific selection; (2) sequence variants in MSY occur with a 1.98-fold higher number of variants than other chromosomes in seven sequenced rat strains; (3) Sry, the most studied MSY gene, has undergone extensive gene duplications, driving ubiquitous expression not seen in human or mouse; (4) the expression profile of Sry in the rat is driven by the insertion of the Sry2 copy into an intron of the ubiquitously expressed Kdm5d gene in antisense orientation, but due to several loss of function mutations in the Sry2 protein, nuclear localization and transcriptional control are decreased; (5) expression of Sry copies other than Sry2 in the rat overlaps with the expression profile for human SRY; (6) gene duplications and sequence variants (P76T) of Sry can be selected for phenotypes such as high blood pressure and androgen receptor signaling within inbred mating; and most importantly, (7) per chromosome size, MSY contributes to higher strain-specific phenotypic variation relative to all other chromosomes, with 53 phenotypes showing both a male to female and consomic cross significance. Conclusion The data presented supports a high probability of MSY genetic variation altering a broad range of inbred rat phenotypes. Electronic supplementary material The online version of this article (doi:10.1186/s13293-016-0064-z) contains supplementary material, which is available to authorized users.

Background The pathological hallmarks of transmissible spongiform encephalopathy (TSE) diseases are the deposition of a misfolded form of a host-encoded protein (PrPres), marked astrocytosis, microglial activation and spongiosis. The development of powerful gene based technologies has permitted increased levels of pro-inflammatory cytokines to be demonstrated. However, due to the use of assays of differing sensitivities and typically the analysis of a single model system it remained unclear whether this was a general feature of these diseases or to what extent different model systems and routes of infection influenced the relative levels of expression. Similarly, it was not clear whether the elevated levels of cytokines observed in the brain were accompanied by similar increases in other tissues that accumulate PrPres, such as the spleen. Results The level of expression of the three interferon responsive genes, Eif2ak2, 2'5'-OAS, and Mx2, was measured in the brains of Syrian hamsters infected with scrapie 263K, VM mice infected with bovine spongiform encephalopathy and C57BL/6 mice infected with the scrapie strain ME7. Glial fibrillary acidic expression confirmed the occurrence of astrocytosis in all models. When infected intracranially all three models showed a similar pattern of increased expression of the interferon responsive genes at the onset of clinical symptoms. At the terminal stage of the disease the level and pattern of expression of the three genes was mostly unchanged in the mouse models. In contrast, in hamsters infected by either the intracranial or intraperitoneal routes, both the level of expression and the expression of the three genes relative to one another was altered. Increased interferon responsive gene expression was not observed in a transgenic mouse model of Alzheimer's disease or the spleens of C57BL/6 mice infected with ME7. Concurrent increases in TNFα, TNFR1, Fas/ApoI receptor, and caspase 8 expression in ME7 infected C57BL/6 mice were observed. Conclusion The identification of increased interferon responsive gene expression in the brains of three rodent models of TSE disease at two different stages of disease progression suggest that this may be a general feature of the disease in rodents. In addition, it was determined that the increased interferon responsive gene expression was confined to the CNS and that the TSE model system and the route of infection influenced the pattern and extent of the increased expression. The concurrent increase in initiators of Eif2ak2 mediated apoptotic pathways in C57BL/6 mice infected with ME7 suggested one mechanism by which increased interferon responsive gene expression may enhance disease progression.