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Background In chronic lymphocytic leukemia, a better understanding of leukemic cell characteristics after treatment would help to design specific therapeutic approaches aimed at preventing clinical relapse. Gene arrays have become a powerful approach to perform gene expression profiling; nevertheless, to work with residual cells entails an intensive labor. The aim of this study was to set forth an effective gene expression approach to analyze residual leukemic cells. Methods Leukocytes from CLL patient's samples were sorted by flow cytometry using a 6-color panel. The quality and quantity of RNA isolated from different inputs of cells were compared by two silica column protocols: RNeasy Micro and RNeasy Mini. RNA amplifications were carried out according to two manufacturer's protocols: Ovation Pico SL and Ovation Pico WTA. A total of 3.5 mu g of cDNA was labeled and hybridized to Human Gene 2.0 ST arrays. Results RNA extracted from low number of input cells by RNeasy Micro showed similar RNA integrity number to that obtained from RNeasy Mini; however, the RNA quantity was higher using the RNeasy Micro Kit. In addition, those RNA samples obtained with RNeasy Micro and amplified with Ovation Pico WTA showed good quality to proceed for a gene array study, independently of the number of input cells (range: 1 x 10(4)-5 x 10(5) cells). Conclusions We observed that this workflow is a feasible approach to obtain genomic material extracted from leukemic cells as little as 1 x 10(4) cells and it can be useful to carry out gene expression profile experiments to characterize residual leukemic cells in chronic lymphocytic leukemia.

Ibrutinib, an oral inhibitor of Bruton's tyrosine kinase (BTK), at a once-daily dose of 420 mg achieved BTK active-site occupancy in patients with chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) that was maintained at 24 hours. It is unknown if intermittent interruption of ibrutinib therapy contributes to altered clinical outcomes. We therefore evaluated the effect of ibrutinib dose adherence on patient outcomes in the phase 3 RESONATE trial. The overall mean dose intensity (DI) was 95% with median treatment duration of similar to 9 months. Pharmacokinetic assessment of ibrutinib exposure at 420-mg dose suggested similar exposure regardless of patient weight or age. As assessed by independent review committee, patients with higher DI experienced longer median progression-free survival (PFS) compared with those with lower DI regardless of del17p and/or TP53 status. Of 79 patients requiring a drug hold, treatment was restarted at the original dose in 73 (92%) patients. Mean duration of a missed-dose event was 18.7 days (range, 8-56). Patients missing >= 8 consecutive days of ibrutinib had a shorter median PFS vs those missing

In normal B-cells, B-cell antigen receptor (BCR) signaling can be negatively regulated by the low-affinity receptor FcγRIIb (CD32b). To better understand the role of FcγRIIb in chronic lymphocytic leukemia (CLL), we correlated its expression on 155 samples from newly-diagnosed Binet A patients with clinical characteristics and outcome. FcγRIIb expression was similar in normal B-cells and leukemic cells, this being heterogenous among patients and within CLL clones. FcγRIIb expression did not correlate with well known prognostic markers [disease stage, serum beta-2 microglobulin (B2M), IGHV mutational status, expression of ZAP-70 and CD38, and cytogenetics] except for a weak concordance with CD49d Moreover, patients with low FcγRIIb expression (69/155, 44.5%) required therapy earlier than those with high FcγRIIb expression (86/155, 55.5%) (median 151.4 months vs. not reached; p=0.071). These results encourage further investigation on the role of FcγRIIb in CLL biology and prognostic significance in larger series of patients.

The majority of causative variants in familial breast cancer remain unknown. Of the known risk variants, most are tumor cell autonomous, and little attention has been paid yet to germline variants that may affect the tumor microenvironment. In this study, we developed a system called the Consomic Xenograft Model (CXM) to map germline variants that affect only the tumor microenvironment. In CXM, human breast cancer cells are orthotopically implanted into immunodeficient consomic strains and tumor metrics are quantified (e.g., growth, vasculogenesis, and metastasis). Because the strain backgrounds vary, whereas the malignant tumor cells do not, any observed changes in tumor progression are due to genetic differences in the nonmalignant microenvironment. Using CXM, we defined genetic variants on rat chromosome 3 that reduced relative tumor growth and hematogenous metastasis in the SS.BN3IL2Rγ consomic model compared with the SSIL2Rγ parental strain. Paradoxically, these effects occurred despite an increase in the density of tumor-associated blood vessels. In contrast, lymphatic vasculature and lymphogenous metastasis were unaffected by the SS.BN3IL2Rγ background. Through comparative mapping and whole-genome sequence analysis, we narrowed candidate variants on rat chromosome 3 to six genes with a priority for future analysis. Collectively, our results establish the utility of CXM to localize genetic variants affecting the tumor microenvironment that underlie differences in breast cancer risk. Cancer Res; 74(22); 6419–29. ©2014 AACR.

Genome-wide association studies (GWAS) are useful for nominating candidate genes, but typically are unable to establish disease causality or differentiate between the effects of variants in linkage disequilibrium (LD). Additionally, some GWAS loci might contain multiple causative variants or genes that contribute to the overall disease susceptibility at a single locus. However, the majority of current GWAS lack the statistical power to test whether multiple causative genes underlie the same locus, prompting us to adopt an alternative approach to testing multiple GWAS genes empirically. We used gene targeting in a disease-susceptible rat model of genetic hypertension to test all six genes at the Agtrap-Plod1 locus (Agtrap, Mthfr, Clcn6, Nppa, Nppb, and Plod1) for blood pressure (BP) and renal phenotypes. This revealed that the majority of genes at this locus (five out of six) can impact hypertension by modifying BP and renal phenotypes. Mutations of Nppa, Plod1, and Mthfr increased disease susceptibility, whereas Agtrap and Clcn6 mutations decreased hypertension risk. Reanalysis of the human AGTRAP-PLOD1 locus also implied that disease-associated haplotype blocks with polygenic effects were not only possible, but rather were highly plausible. Combined, these data demonstrate for the first time that multiple modifiers of hypertension can cosegregate at a single GWAS locus.

Renin was the first blood pressure (BP) quantitative trait locus mapped by linkage analysis in the rat. Subsequent BP linkage and congenic studies capturing different portions of the renin region have returned conflicting results, suggesting that multiple interdependent BP loci may be residing in the chromosome 13 BP quantitative trait locus that includes Renin . We used SS-13 BN congenic strains to map 2 BP loci in the Renin region (chr13: 45.2–49.0 Mb). We identified a 1.1-Mb protective Brown Norway region around Renin (chr13: 46.1–47.2 Mb) that significantly decreased BP by 32 mm Hg. The Renin protective BP locus was offset by an adjacent hypertensive locus (chr13: 47.2–49.0 Mb) that significantly increased BP by 29 mm Hg. Sequence analysis of the protective and hypertensive BP loci revealed 1433 and 2063 variants between Dahl salt-sensitive/Mcwi and Brown Norway rats, respectively. To further reduce the list of candidate variants, we regenotyped an overlapping SS-13 SR congenic strain (S/ren rr ) with a previously reported BP phenotype. Sequence comparison among Dahl salt-sensitive, Dahl R, and Brown Norway reduced the number of candidate variants in the 2 BP loci by 42% for further study. Combined with previous studies, these data suggest that at least 4 BP loci reside within the 30-cM chromosome 13 BP quantitative trait locus that includes Renin .

We previously reported that the fawn-hooded hypertensive (FHH) rat is a natural Rab38 knockout, supported by a congenic animal (FHH.BN-Rab38) having less proteinuria than FHH animals. Because these congenic animals contain Brown Norway (BN) alleles for five other named genes; however, a causal role for Rab38 in the FHH phenotype remains uncertain. Here, we used transgenic and knockout models to validate Rab38 and to exclude other genes within the 1.5 Mb congenic region from involvement in causing the FHH phenotype. Transgenic rats homozygous for the wild-type Rab38 BN allele on the FHH background exhibited phenotypic rescue, having 43% lower proteinuria and 75% lower albuminuria than nontransgenic FHH littermates. Conversely, knockout of the Rab38 gene on the FHH.BN-Rab38 congenic line recapitulated a proteinuric phenotype indistinguishable from the FHH strain. In addition, in cultured proximal tubule LLC-PK1 cells, knockdown of Rab38 mRNA significantly decreased endocytosis of colloidal gold-coupled albumin, supporting the hypothesis that Rab38 modulates proteinuria through effects on tubular re-uptake and not by altering glomerular permeability. Taken together, these findings validate Rab38 as a gene having a causal role in determining the phenotype of the FHH rat, which models hypertension-associated renal disease. Furthermore, our data suggest that Rab38 affects urinary protein excretion via effects in the proximal tubule.

Objective:The Dahl salt-sensitive rat is a well-established model of salt-sensitive hypertension. The goal of this study was to assess the expression and activity of renal sodium channels and transporters in the renin-deficient salt-sensitive rat.Methods:Renin knockout (Ren−/−) rats created on the salt-sensitive rat background were used to investigate the role of renin in the regulation of ion transport in salt-sensitive hypertension. Western blotting and patch-clamp analyses were utilized to assess the expression level and activity of Na+transporters.Results:It has been described previously that Ren−/−rats exhibit severe kidney underdevelopment, polyuria, and lower body weight and blood pressure compared to their wild-type littermates. Here we found that renin deficiency led to decreased expression of sodium-hydrogen antiporter (NHE3), the Na+/H+exchanger involved in Na+absorption in the proximal tubules, but did not affect the expression of Na-K-Cl cotransporter (NKCC2), the main transporter in the loop of Henle. In the distal nephron, the expression of sodium chloride cotransporter (NCC) was lower in Ren−/−rats. Single-channel patch clamp analysis detected decreased ENaC activity in Ren−/−rats which was mediated via changes in the channel open probability.Conclusion:These data illustrate that renin deficiency leads to significant dysregulation of ion transporters.

Germline transgenesis is an important procedure for functional investigation of biological pathways, as well as for animal biotechnology. We have established a simple, nonviral protocol in three important biomedical model organisms frequently used in physiological studies. The protocol is based on the hyperactive Sleeping Beauty transposon system, SB100X, which reproducibly promoted generation of transgenic founders at frequencies of 50–64, 14–72, and 15% in mice, rats, and rabbits, respectively. The SB100X-mediated transgene integrations are less prone to genetic mosaicism and gene silencing as compared to either the classical pronuclear injection or to lentivirus-mediated transgenesis. The method was successfully applied to a variety of transgenes and animal models, and can be used to generate founders with single-copy integrations. The transposon vector also allows the generation of transgenic lines with tissue-specific expression patterns specified by promoter elements of choice, exemplified by a rat reporter strain useful for tracking serotonergic neurons. As a proof of principle, we rescued an inborn genetic defect in the fawn-hooded hypertensive rat by SB100X transgenesis. A side-by-side comparison of the SB100X- and piggyBac-based protocols revealed that the two systems are complementary, offering new opportunities in genome manipulation.—Katter, K., Geurts, A. M., Hoffmann, O., Mátés, L., Landa,V., Hiripi, L., Moreno, C., Lazar, J., Bashir, S., Zidek, V., Popova, E., Jerchow, B., Becker, K., Devaraj, A., Walter, I., Grzybowksi, M., Corbett, M., Rangel Filho, A., Hodges, M. R., Bader, M., Ivics, Z., Jacob, H. J., Pravenec, M., Bősze, Z., Rülicke, T., Izsvák, Z. Transposon-mediated transgenesis, transgenic rescue, and tissue-specific gene expression in rodents and rabbits.

The majority of ESRD cases are associated with diabetes, hypertension, or both; however, numerous studies in both humans and animal models suggest that renal disease susceptibility genes exist that are independent of the initiating factor.1–6 The dissection of quantitative traits for CKD in humans remains challenging due to heterogeneity and environmental variability.7,8 Consequently, there is a need to pursue other strategies for identifying genes and their associated pathways that are driving CKD. One solution is to use congenic rat models to investigate regions of the rat genome that are responsible for renal impairment. One of the first examples of genetic dissection of renal impairment in rats was demonstrated in F2 crosses between the renal disease–susceptible Fawn-hooded hypertensive (FHH) rat and the renal disease–resistant August Copenhagen Irish (ACI) rat.9,10 These studies led to the identification of five quantitative trait loci (QTLs) linked to the severity of proteinuria called Renal Failure-1 through -5 (Rf-1 through -5).9,10 Gene–gene interactions were found between the various Rf QTLs, because the presence of homozygous FHH alleles in multiple Rf QTLs resulted in a synergistic increase in proteinuria severity.10 An interaction was specifically identified between Rf-1 and Rf-4, located on rat chromosome 1 and 14, respectively. Van Dijk et al. generated single (Rf-1a and Rf-4) and double (Rf-1a+4) congenic animals, and found that the Rf-1a and Rf-4 single congenic animals did not show increased proteinuria compared with the ACI control strain. Only the transfer of both Rf-1a and Rf-4, as in the Rf-1a+4 double congenic strain, conferred a significant increase in proteinuria.11 The original Rf-4 region consisted of 61.9 Mb containing 499 known and predicted genes. To narrow the region of interest to begin a meaningful search for the causal variant, it is necessary to physically reduce the candidate region in turn reducing the number of candidate genes. In this study, we use congenic mapping to generate a minimal congenic line called Rf-1a+4_a, which carries only 4.1 Mbp of FHH genome in the Rf-4 region that we show significantly contributes to proteinuria. Van Dijk et al. previously demonstrated that genes in the Rf-1 region increase glomerular capillary pressure (PGC) by impairing renal blood flow autoregulation.11 To explain the interaction between Rf-4 and Rf-1, they hypothesized that the Rf-4 region affects integrity of the glomerular filtration barrier that is manifested when exposed to an increase in PGC (i.e., Rf-1).12 Here, we assessed glomerular permeability to albumin (Palb) in the Rf-1a+4_a congenics to address this hypothesis, and found that Palb was significantly higher in Rf-1a+4_a compared with control strains. The refined interval is only 6.6% of the original Rf-4 congenic region containing just 67 known and predicted genes. To initiate the search for causative variants, we analyzed the genomic sequence of the entire congenic region. Within the coding sequence, we found only one benign nonsynonymous amino acid variant between ACI and FHH in the entire Rf-4_a region, suggesting that an intergenic, intronic, or untranslated variant(s) is likely responsible for the Rf-4_a renal phenotype. It has been demonstrated that conserved sequences suggest functionality,13–15 so we prioritized noncoding variants based on evolutionary conservation. Using a congenic model and comparative genomic approach, we have reduced the candidates for the Rf-4 QTL to a handful of sequence variants. The results of this study are of particular interest because previous studies have indicated that the homologous region on human chromosome 4 is associated with various forms of CKD as indicated by both linkage16 and genome-wide association studies (GWASs).17,18

Impaired regulation of renin in Dahl salt-sensitive rats (SS/JRHsdMcwi, SS) contributes to attenuated angiogenesis in this strain. This study examined angiogenic function and genomic structure of regions surrounding the renin gene using subcongenic strains of the SS and BN/NHsdMcwi (BN) rat to identify important genomic variations between SS and BN involved in angiogenesis. Three candidate regions on Chr 13 were studied: two congenic strains containing 0.89 and 2.62 Mb portions of BN Chr 13 that excluded the BN renin allele and a third strain that contained a 2.02 Mb overlapping region that included the BN renin allele. Angiogenesis induced by electrical stimulation of the tibialis anterior muscle was attenuated in the SS compared with the BN. Congenics carrying the SS renin allele had impaired angiogenesis, while strains carrying the BN renin allele had angiogenesis restored. The exception was a congenic including a region of BN genome 0.4 Mb distal to renin that restored both renin regulation and angiogenesis. This suggests that there is a distant regulatory element in the BN capable of restoring normal regulation of the SS renin allele. The importance of ANG II in the restored angiogenic response was demonstrated by blocking with losartan. Sequencing of the 4.05 Mb candidate region in SS and BN revealed a total of 8,850 SNPs and other sequence variants. An analysis of the genes and their variants in the region suggested a number of pathways that may explain the impaired regulation of renin and angiogenesis in the SS rat.

The renin-angiotensin system plays an important role in the control of blood pressure (BP) and renal function. To illuminate the importance of renin in the context of a disease background in vivo, we used zinc-finger nucleases (ZFNs) designed to target the renin gene and create a renin knockout in the SS/JrHsdMcwi (SS) rat. ZFN against renin caused a 10-bp deletion in exon 5, resulting in a frameshift mutation. Plasma renin activity was undetectable in the Ren −/− rat, and renin protein was absent from the juxtaglomerular cells in the kidney. Body weight was lower in the Ren −/− rats (than in the Ren +/− or wild-type littermates), and conscious BP on low-salt diet (0.4% NaCl) was 58±2 mm Hg in the Ren −/− male rats versus 117 mm Hg in the Ren +/− littermates, a reduction of almost 50 mm Hg. Blood urea nitrogen (BUN) and plasma creatinine levels were elevated in the Ren −/− strain (BUN 112±7 versus 23±2 mg/dL and creatinine 0.53±0.02 versus 0.26±0.02 mg/dL), and kidney morphology was abnormal with a rudimentary inner renal medulla, cortical interstitial fibrosis, thickening of arterial walls, and abnormally shaped glomeruli. The development of the first rat knockout in the renin-angiotensin system demonstrates the efficacy of the ZFN technology for creating knockout rats for cardiovascular disease on any genetic background and emphasizes the role of renin in BP regulation and kidney function even in the low-renin SS rat.

This study evaluated endothelium-dependent vascular relaxation in response to acetylcholine (ACh) in isolated middle cerebral arteries (MCA) from Dahl salt-sensitive (Dahl SS) rats and three different congenic strains that contain a portion of Brown Norway (BN) chromosome 13 introgressed onto the Dahl SS genetic background through marker-assisted breeding. Two of the congenic strains carry a 3.5-Mbp portion and a 2.6-Mbp portion of chromosome 13 that lie on opposite sides of the renin locus, while the third contains a 2.0-Mbp overlapping region that includes the BN renin allele. While maintained on a normal salt (0.4% NaCl) diet, MCAs from Dahl SS rats and the congenic strains retaining the Dahl SS renin allele failed to dilate in response to ACh, whereas MCAs from the congenic strain carrying the BN renin allele exhibited normal vascular relaxation. In congenic rats receiving the BN renin allele, vasodilator responses to ACh were eliminated by nitric oxide synthase inhibition with NG-nitro-l-arginine methyl ester, angiotensin-converting enzyme inhibition with captopril, and AT1 receptor blockade with losartan. NG-nitro-l-arginine methyl ester-sensitive vasodilation in response to ACh was restored in MCAs of Dahl SS rats that received either a 3-day infusion of a subpressor dose of angiotensin II (3 ng·kg−1·min−1 iv), or chronic treatment with the superoxide dismutase mimetic tempol (15 mg·kg−1·day−1). These findings indicate that the presence of the Dahl SS renin allele plays a crucial role in endothelial dysfunction present in the cerebral circulation of the Dahl SS rat, even in the absence of elevated dietary salt intake, and that introgression of the BN renin allele rescues endothelium-dependent vasodilator responses by restoring normal activation of the renin-angiotensin system.

The rat has long been a model favored by physiologists, pharmacologists and neuroscientists. However, over the past two decades, many investigators in these fields have turned to the mouse because of its gene modification technologies and extensive genomic resources. Although the genomic resources of the rat have nearly caught up, gene targeting has lagged far behind, limiting the value of the rat for many investigators. In the past two years, advances in transposon- and zinc finger nuclease (ZFN)-mediated gene knockout as well as the establishment and culturing of embryonic and inducible pluripotent stem cells have created new opportunities for rat genetic research. Here, we provide a high-level description and the potential uses of these new technologies for investigators using the rat for biomedical research.

Congenic strains continue to be a fundamental resource for dissecting the genetic basis of complex traits. Traditionally, genetic variants (QTLs) that account for phenotypic variation in a panel of congenic strains are sought first by comparing phenotypes for each strain to the host (reference) strain, and then by examining the results to identify a common chromosome segment that provides the best match between genotype and phenotype across the panel. However, this ‘‘common-segment’’ method has significant limitations, including the subjective nature of the genetic model and an inability to deal formally with strain phenotypes that do not fit the model. We propose an alternative that we call ‘‘sequential’’ analysis and that is based on a unique principle of QTL analysis where each strain, corresponding to a single genotype, is tested individually for QTL effects rather than testing the congenic panel collectively for common effects across heterogeneous backgrounds. A minimum spanning tree, based on principles of graph theory, is used to determine the optimal sequence of strain comparisons. For two traits in two panels of congenic strains in mice, we compared results for the sequential method with the common-segment method as well as with two standard methods of QTL analysis, namely, interval mapping and multiple linear regression. The general utility of the sequential method was demonstrated with analysis of five additional traits in congenic panels from mice and rats. Sequential analysis rigorously resolved phenotypic heterogeneity among strains in the congenic panels and found QTLs that other methods failed to detect.

Clonal evolution and outgrowth of cellular variants with additional chromosomal abnormalities are major causes of disease progression in chronic lymphocytic leukemia (CLL). Because new DNA lesions occur during S phase, proliferating cells are at the core of this problem. In this study, we used in vivo deuterium (2H) labeling of CLL cells to better understand the phenotype of proliferating cells in 13 leukemic clones. In each case, there was heterogeneity in cellular proliferation, with a higher fraction of newly produced CD38+ cells compared with CD38− counterparts. On average, there were 2-fold higher percentages of newly born cells in the CD38+ fraction than in CD38− cells; when analyzed on an individual patient basis, CD38+2H-labeled cells ranged from 6.6% to 73%. Based on distinct kinetic patterns, interclonal heterogeneity was also observed. Specifically, 4 patients exhibited a delayed appearance of newly produced CD38+ cells in the blood, higher leukemic cell CXC chemokine receptor 4 (CXCR4) levels, and increased risk for lymphoid organ infiltration and poor outcome. Our data refine the proliferative compartment in CLL based on CD38 expression and suggest a relationship between in vivo kinetics, expression of a protein involved in CLL cell retention and trafficking to solid tissues, and clinical outcome.

Chronic lymphocytic leukemia (CLL) is uniquely characterized by the existence of subsets of cases with quasi-identical, 'stereotyped' B-cell receptors (BCRs). Herein we investigate this stereotypy in 2662 patients with CLL, the largest series yet, using purpose-built bioinformatics methods based on sequence pattern discovery. Besides improving the identification of 'stereotyped' cases, we demonstrate that CLL actually consists of two different categories, based on the BCR repertoire, with important biological and ontogenetic differences. The first (similar to 30% of cases) shows a very restricted repertoire and is characterized by BCR stereotypy (clustered cases), whereas the second includes cases with heterogeneous BCRs (nonclustered cases). Eleven major CLL clusters were identified with antigen-binding sites defined by just a few critically positioned residues, regardless of the actual immunoglobulin (IG) variable gene used. This situation is closely reminiscent of the receptors expressed by cells participating in innate immune responses. On these grounds, we argue that whereas CLL cases with heterogeneous BCRs likely derive from the conventional B-cell pool, cases with stereotyped BCRs could derive from progenitor cells evolutionarily adapted to particular antigenic challenges, perhaps intermediate between a true innate immune system and the conventional adaptive B-cell immune system, functionally similar to what has been suggested previously for mouse B1 cells. Leukemia (2010) 24, 125-132; doi:10.1038/leu.2009.186; published online 17 September 2009

The combination of the neoplastic accumulation of mature B lymphocytes with the presence of autoimmune phenomena is a characteristic finding in chronic B cell lymphoproliferative disorders, particularly chronic lymphocytic leukemia. Identification of mechanisms linking neoplasia to the autoimmune defects is important for a better understanding and improving the treatment of these conditions. Among such mechanisms, the B cell activator factor (BAFF) and a proliferation-inducing ligand (APRIL), two members of the tumor necrosis factor family, play an important role. BAFF and APRIL have both been associated with autoimmunity, with their underlying mechanism of action most likely being related to the rescue of autoreactive B cells. In addition, BAFF and APRIL are crucial in B cell development and homeostasis particularly via the activation of NF-kappaB pathway-mediated survival signals. These two proteins, therefore, constitute a paradigm of pathophysiological defects linking neoplasia and autoimmunity, thereby providing a better understanding of chronic B cell lymphoproliferative disorders.

This study examined the effect of transfer of overlapping regions of chromosome 5 that includes (4A+) or excludes (4A−) the cytochrome P-450 4A (CYP4A) genes from the Lewis rat on the renal production of 20-hydroxyeicosatetraenoic acid (20-HETE) and the development of hypertension-induced renal disease in congenic strains of Dahl salt-sensitive (Dahl S) rats. The production of 20-HETE was higher in the outer medulla of 4A+than in Dahl S or 4A−rats. Mean arterial pressure (MAP) rose to 190 ± 7 and 185 ± 3 mmHg in Dahl S and 4A−rats fed a high-salt (HS) diet for 21 days but only to 150 ± 5 mmHg in the 4A+strain. Protein excretion increased to 423 ± 40 and 481 ± 37 mg/day in Dahl S and 4A−rats vs. 125 ± 15 mg/day in the 4A+strain. Baseline glomerular capillary pressure (Pgc) was lower in 4A+rats (38 ± 1 mmHg) than in Dahl S rats (42 ± 1 mmHg). Pgc increased to 50 ± 1 mmHg in Dahl S rats fed a HS diet, whereas it remained unaltered in 4A+rats (39 ± 1 mmHg). Baseline glomerular permeability to albumin ( Palb) was lower in 4A+rats (0.19 ± 0.05) than in Dahl S or 4A−rats (0.39 ± 0.02). Palbrose to ∼0.61 ± 0.03 in 4A−and Dahl S rats fed a HS diet for 7 days, but it remained unaltered in the 4A+rats. The expression of transforming growth factor-β2 was higher in glomeruli of Dahl S rats than in 4A+rats fed either a low-salt (LS) or HS diet. Chronic administration of a 20-HETE synthesis inhibitor (HET0016; 10 mg·kg−1·day−1sc) reversed the fall in MAP and renoprotection seen in 4A+rats. These results indicate that the introgression of the CYP4A genes from Lewis rats into the Dahl S rats increases the renal formation of 20-HETE and attenuates the development of hypertension and renal disease.

To determine the independent and combined effects of three quantitative trait loci (QTL) for blood pressure in the Genetically Hypertensive (GH/Omr) rat by generating and characterizing single and combined congenic strains that have QTL on rat chromosomes (RNO) 2, 6, and 18 from the GH rat introduced into a hypertension resistant Brown Norway (BN) background.Linkage analysis and QTL identification (genome wide QTL scan) were performed with MapMaker/EXP to build the genetic maps and MapMaker/QTL for linking the phenotypes to the genetic map. The congenic strains were derived using marker-assisted selection strategy from a single male F1 offspring of an intercross between the male GH/Omr and female BN/Elh, followed by 10 generations of selective backcrossing to the female BN progenitor strain. Single congenic strains generated were BN.GH-(D2Rat22-D2Mgh11)/Mcwi (BN.GH2); BN.GH-(D6Mit12-D6Rat15)/Mcwi (BN.GH6); and BN.GH-(D18Rat41-D18Mgh4)/Mcwi (BN.GH18). Blood pressure measurements were obtained either via a catheter placed in the femoral artery or by radiotelemetry. Responses to angiotensin II (ANGII), norepinephrine (NE), and baroreceptor sensitivity were measured in the single congenics.Transferring one or more QTL from the hypertensive GH into normotensive BN strain was not sufficient to cause hypertension in any of the developed congenic strains. There were no differences between the parental and congenic strains in their response to NE. However, BN.GH18 rats revealed significantly lower baroreceptor sensitivity (beta=-1.25-/+0.17), whereas BN.GH2 (beta=0.66-/+0.09) and BN.GH18 (beta=0.71-/+0.07) had significantly decreased responses to ANGII from those observed in the BN (beta=0.88-/+0.08).The failure to alter blood pressure levels by introducing the hypertensive QTL from the GH into the hypertension resistant BN background suggests that the QTL effects are genome background-dependent in the GH rat. BN.GH2 and BN.GH18 rats reveal significant differences in response to ANGII and impaired baroreflex sensitivity, suggesting that we may have captured a locus responsible for the genetic control of baroreceptor sensitivity, which would be considered an intermediate phenotype of blood pressure.

Previous studies have indicated the importance of angiotensin II (ANG II) in skeletal muscle angiogenesis. The present study explored the effect of regulation of the renin gene on angiogenesis induced by electrical stimulation with the use of physiological, pharmacological, and genetic manipulations of the renin-angiotensin system (RAS). Transfer of the entire chromosome 13, containing the physiologically regulated renin gene, from the normotensive inbred Brown Norway (BN) rat into the background of an inbred substrain of the Dahl salt-sensitive (SS/Mcwi) rat restored renin levels and the angiogenic response after electrical stimulation. This restored response was significantly attenuated when SS-13BN/Mcwi consomic rats were treated with lisinopril or high-salt diet. The role of ANG II on this effect was confirmed by the complete restoration of skeletal muscle angiogenesis in SS/Mcwi rats infused with subpressor doses of ANG II. Congenic strains derived from the SS-13BN/Mcwi consomic were used to further verify the role of the renin gene in this response. Microvessel density was markedly increased after stimulation in congenic strains that contained the renin gene from the BN rat (congenic lines A and D). This angiogenic response was suppressed in control strains that carried regions of the BN genome just above (congenic line C) or just below (congenic line B) the renin gene. The present study emphasizes the importance of maintaining normal renin regulation as well as ANG II levels during the angiogenesis process with a combination of physiological, genetic, and pharmacological manipulation of the RAS.

Somatic hypermutation (SHM) features in a series of 1967 immunoglobulin heavy chain gene (IGH) rearrangements obtained from patients with chronic lymphocytic leukemia (CLL) were examined and compared with IGH sequences from non-CLL B cells available in public databases. SHM analysis was performed for all 1290 CLL sequences in this cohort with less than 100% identity to germ line. At the cohort level, SHM patterns were typical of a canonical SHM process. However, important differences emerged from the analysis of certain subgroups of CLL sequences defined by: (1) IGHV gene usage, (2) presence of stereotyped heavy chain complementarity-determining region 3 (HCDR3) sequences, and (3) mutational load. Recurrent, “stereotyped” amino acid changes occurred across the entire IGHV region in CLL subsets carrying stereotyped HCDR3 sequences, especially those expressing the IGHV3-21 and IGHV4-34 genes. These mutations are underrepresented among non-CLL sequences and thus can be considered as CLL-biased. Furthermore, it was shown that even a low level of mutations may be functionally relevant, given that stereotyped amino acid changes can be found in subsets of minimally mutated cases. The precise targeting and distinctive features of somatic hypermutation (SHM) in selected subgroups of CLL patients provide further evidence for selection by specific antigenic element(s).

Regulation of medullary blood flow (MBF) is essential in maintaining renal function and blood pressure. However, it is unknown whether outer MBF (OMBF) and papillary blood flow (PBF) are regulated independently when extracellular volume (ECV) is enhanced. The aim of this study was to determine whether OMBF and PBF are differently regulated and whether there is an interaction between nitric oxide (NO), prostaglandins (PGs) and angiotensin II (Ang II) in regulating OMBF and PBF when ECV is enhanced. To achieve these goals, OMBF and PBF were measured by laser-Doppler in volume-expanded rats treated with a cyclooxygenase inhibitor (meclofenamate, 3 mg/kg) and/or a NO synthesis inhibitor (L-nitro-arginine methyl ester (L-NAME), 3 μg/kg/min) and/or Ang II (10 ng/kg/min). OMBF was unchanged by NO or PGs synthesis inhibition but decreased by 36 % (P

The present study evaluated whether the impairment in autoregulation of renal blood flow (RBF) in the fawn-hooded Hypertensive (FHH) rat colocalizes with the Rf-1 region on chromosome 1 that has been previously linked to the development of proteinuria in this strain. Autoregulation of RBF was measured in FHH and a consomic strain (FHH.1BN) in which chromosome 1 from the Brown-Norway (BN) rat was introgressed into the FHH genetic background. The autoregulation indexes (AI) averaged 0.80 ± 0.08 in the FHH and 0.19 ± 0.05 in the FHH.1BNrats. We next performed a genetic linkage analysis for autoregulation of RBF in 85 F2 rats generated from a backcross of FHH.1BNconsomic and FHH rats. The results revealed a significant quantitative trait locus (QTL) with a peak logarithm of the odds score of 6.3 near marker D1Rat376. To confirm the existence of this QTL, five overlapping congenic strains were created that spanned the region from markers D1Rat234 to D1Mit14. Transfer of a region of BN chromosome 1 from markers D1Mgh13 to D1Rat89 into the FHH genetic background improved autoregulation of RBF (AI = 0.23 ± 0.04) and reduced protein excretion. In contrast, RBF was poorly autoregulated and the rats were not protected from proteinuria in congenic strains in which other regions of chromosome 1 that exclude the D1Rat376 marker were transferred. These results indicate that there is a gene(s) that influences autoregulation of RBF and proteinuria between markers D1Mgh13 and D1Rat89 on chromosome 1 that lies within the confidence interval of the Rf-1 QTL previously linked to the development of proteinuria in FHH rats.

The need to translate genes to function has positioned the rat as an invaluable animal model for genomic research. The significant increase in genomic resources in recent years has had an immediate functional application in the rat. Many of the resources for translational research are already in place and are ready to be combined with the years of physiological knowledge accumulated in numerous rat models, which is the subject of this perspective. Based on the successes to date and the research projects under way to further enhance the infrastructure of the rat, we also project where research in the rat will be in the near future. The impact of the rat genome project has just started, but it is an exciting time with tremendous progress.

ZAP-70 is a tyrosine kinase that participates in early B-cell differentiation and is a prognostic factor in chronic lymphocytic leukaemia (CLL), where it is associated with an unmutated configuration of the IgV(H) genes. In this study ZAP-70 expression was studied by immunohistochemistry in a spectrum of B-cell lymphoid neoplasms; this staining method was compared with flow cytometry, and the relationship of ZAP-70 expression to mutational status and prognosis was assessed. 242 tissue samples from 225 patients with B-cell lymphoid neoplasms arising at different maturational stages were included. Flow cytometry was performed in all CLL cases (n = 52). IgV(H) mutational status was determined in 25 CLL and 12 mantle cell lymphoma (MCL) patients. ZAP-70 was positive in 34/52 (65%) CLL, 9/31 (31%) Burkitt's lymphoma, 2/7 (29%) lymphoblastic lymphomas, 3/36 (8%) MCL, 1/23 (4%) marginal zone lymphoma, and 1/45 (2%) diffuse large B-cell lymphomas, but in none of the 19 follicular lymphomas or the 14 Hodgkin lymphomas. An identical ZAP-70 pattern was obtained in six patients with simultaneous biopsies from different sites and in 12 patients with sequential biopsies. Immunohistochemistry and flow cytometry gave identical results in 48 the 52 CLLs. All but one ZAP-70-positive CLL had IgV(H) gene in an unmutated configuration, whereas all but one ZAP-70-negative CLL had somatically hypermutated IgV(H). The 12 MCLs analysed were ZAP-70 negative regardless of IgV(H) mutational status (4 mutated, 8 unmutated). ZAP-70 positive CLL was associated with a shorter overall survival (median time 103 months vs. 293 months, p = 0.01) and a shorter time to disease progression (median time 26 months vs. 60 months, p = 0.01). In conclusion, ZAP-70 is expressed in several types of B-cell neoplasm and is easily detected by immunohistochemistry, providing a useful prognostic marker in patients with CLL from whom no other material is available or when other techniques for its assessment cannot be performed.

Studies were designed to examine the hypothesis that the renal medulla of Dahl salt-sensitive (Dahl S) rats has a reduced capacity to generate nitric oxide (NO), which diminishes the ability to buffer against the chronic hypertensive effects of small elevations of circulating ANG II. NO synthase (NOS) activity in the outer medulla of Dahl S rats (arginine-citrulline conversion assay) was significantly reduced. This decrease in NOS activity was associated with the downregulation of protein expression of NOS I, NOS II, and NOS III isoforms in this region as determined by Western blot analysis. In anesthetized Dahl S rats, we observed that a low subpressor intravenous infusion of ANG II (5 ng · kg−1 · min−1) did not increase the concentration of NO in the renal medulla as measured by a microdialysis with oxyhemoglobin trapping technique. In contrast, ANG II produced a 38% increase in the concentration of NO (87 ± 8 to 117 ± 8 nmol/l) in the outer medulla of Brown-Norway (BN) rats. The same intravenous dose of ANG II reduced renal medullary blood flow as determined by laser-Doppler flowmetry in Dahl S, but not in BN rats. A 7-day intravenous ANG II infusion at a dose of 3 ng · kg−1 · min−1 did not change mean arterial pressure (MAP) in the BN rats but increased MAP in Dahl S rats from 120 ± 2 to 138 ± 2 mmHg ( P< 0.05). ANG II failed to increase MAP after NO substrate was provided by infusion of l-arginine (300 μg · kg−1 · min−1) into the renal medulla of Dahl S rats. Intravenous infusion ofl-arginine at the same dose had no effect on the ANG II-induced hypertension. These results indicate that an impaired NO counterregulatory system in the outer medulla of Dahl S rats makes them more susceptible to the hypertensive actions of small elevations of ANG II.

PLEKHA7 (pleckstrin homology domain containing family A member 7) has been found in multiple studies as a candidate gene for human hypertension, yet functional data supporting this association are lacking. We investigated the contribution of this gene to the pathogenesis of salt-sensitive hypertension by mutating Plekha7 in the Dahl salt-sensitive (SS/JrHsdMcwi) rat using zinc-finger nuclease technology. After four weeks on an 8% NaCl diet, homozygous mutant rats had lower mean arterial (149 ± 9 mmHg vs. 178 ± 7 mmHg; P < 0.05) and systolic (180 ± 7 mmHg vs. 213 ± 8 mmHg; P < 0.05) blood pressure compared with WT littermates. Albumin and protein excretion rates were also significantly lower in mutant rats, demonstrating a renoprotective effect of the mutation. Total peripheral resistance and perivascular fibrosis in the heart and kidney were significantly reduced in Plekha7 mutant animals, suggesting a potential role of the vasculature in the attenuation of hypertension. Indeed, both flow-mediated dilation and endothelium-dependent vasodilation in response to acetylcholine were improved in isolated mesenteric resistance arteries of Plekha7 mutant rats compared with WT. These vascular improvements were correlated with changes in intracellular calcium handling, resulting in increased nitric oxide bioavailability in mutant vessels. Collectively, these data provide the first functional evidence that Plekha7 may contribute to blood pressure regulation and cardiovascular function through its effects on the vasculature.

The goal of the present study was to narrow a region of chromosome 13 to only several genes and then apply unbiased statistical approaches to identify molecular networks and biological pathways relevant to blood-pressure salt sensitivity in Dahl salt-sensitive (SS) rats. The analysis of 13 overlapping subcongenic strains identified a 1.37 Mbp region on chromosome 13 that influenced the mean arterial blood pressure by at least 25 mmHg in SS rats fed a high-salt diet. DNA sequencing and analysis filled genomic gaps and provided identification of five genes in this region, Rfwd2, Fam5b, Astn1, Pappa2, and Tnr. A cross-platform normalization of transcriptome data sets obtained from our previously published Affymetrix GeneChip dataset and newly acquired RNA-seq data from renal outer medullary tissue provided 90 observations for each gene. Two Bayesian methods were used to analyze the data: 1) a linear model analysis to assess 243 biological pathways for their likelihood to discriminate blood pressure levels across experimental groups and 2) a Bayesian graphical modeling of pathways to discover genes with potential relationships to the candidate genes in this region. As none of these five genes are known to be involved in hypertension, this unbiased approach has provided useful clues to be experimentally explored. Of these five genes, Rfwd2, the gene most strongly expressed in the renal outer medulla, was notably associated with pathways that can affect blood pressure via renal transcellular Na+and K+electrochemical gradients and tubular Na+transport, mitochondrial TCA cycle and cell energetics, and circadian rhythms.

The two tumour necrosis factor family proteins BAFF (TNFSF13B) and APRIL (TNFSF13) and their receptors [BAFF-R (TNFRSF13C), TACI (TNFRSF13B), BCMA (TNFRSF17)] play a critical role in the survival of normal B cells. The sensitivity of normal B cells to BAFF and APRIL can be modulated by signals regulated by their receptors. This modulation, however, has not been extensively investigated in chronic lymphocytic leukaemia (CLL) cells. We evaluated the expression, regulation and signalling of BAFF and APRIL receptors in normal and in CLL cells upon stimulation through CD40+IL4R and BCR. We further analysed the prognostic value of BAFF and APRIL receptors expression in patients with CLL. BCMA expression was significantly higher on CLL cells than on normal B cells. BCR and CD40+IL4R stimulation promoted an increase in TACI and BCMA expression, cell viability and activation in normal B cells. A similar effect was observed in CLL cells after CD40+IL4R but not BCR stimulation. BCMA expression correlated with unmutated IGHV genes, poor-risk cytogenetics, and short progression-free survival. These findings further characterize the link between CD40+IL4R regulatory signals, BAFF, APRIL and their receptors and the survival of leukaemic cells and clinical features of CLL.

The present study evaluated whether inhibition of guanylyl cyclase (GC) with 1H-(1,2,4)oxadiazolo[4,3-a]quinoxaline-1-one (ODQ) and methylene blue (MB) or inhibition of the renal metabolism of arachidonic acid by cytochrome P-450 (CYP450) enzymes with 1-aminobenzotriazole (ABT) and N-hydroxy- N′-(4 butyl-2-methyl phenyl)formamidine (HET0016) alters the renal tubular and vascular effects of a nitric oxide (NO) donor in vivo. Intrarenal infusion of ODQ or MB at a dose of 170 nmol · kg−1 · min−1 lowered renal blood flow (RBF) by 30 and 15%, respectively; glomerular filtration rate (GFR) by 26 and 18%, respectively; and sodium and water excretion by ∼35%. In rats pretreated with nitro-l-arginine methyl ester (37 nmol · kg−1 · min−1) to block the endogenous production of NO, intrarenal infusion of the NO donor S-nitroso- N-acetylcysteine (S-NO-NAC; 50 nmol · kg−1 · min−1) increased RBF (18%), sodium (73%), and water excretion (61%). ODQ or MB administration blocked the effect of S-NO-NAC on RBF but not the diuretic and natriuretic response. Pretreatment of rats with ABT or HET0016 also abolished the renal vasodilatory response to the NO donor and reduced its diuretic and natriuretic effect. These results indicate that both activation of GC and inhibition of CYP450 enzymes contribute to the renal vascular actions of NO, whereas the natriuretic and diuretic actions of NO appear to be largely CYP450 dependent.

Abstract —It has been reported that bradykinin (BK) can induce or activate both cyclooxygenase (COX) isoforms and that the renal effects of BK seem to be mediated by prostaglandins and NO. The first objective of this study was to evaluate the relative contribution of both COX isoforms in mediating the renal response to BK in anesthetized dogs. The second objective was to examine whether COX-2 inhibition potentiates the renal effects induced by NO reduction during BK administration. Intrarenal BK infusion (8 ng · kg −1 · min −1 , n=6) elicited a significant increment in renal blood flow, sodium excretion, urine volume, and the fractional excretion of lithium. COX-2 inhibition (nimesulide, 5 μg · kg −1 · min −1 , n=6) reduced the renal vasodilatation but did not significantly modify the natriuresis or diuresis secondary to BK. Administration of a nonspecific isozyme COX inhibitor (meclofenamate, 5 μg · kg −1 · min −1 ; n=6) did not induce greater effects than those produced by nimesulide. NO synthesis reduction ( N G -nitro- l -arginine methyl ester [L-NAME], 3 μg · kg −1 · min −1 ) prevented the renal vasodilatation and the increment in the fractional excretion of lithium induced by BK but did not affect the natriuretic or diuretic response. Simultaneous nimesulide infusion did not modify the renal effects of L-NAME during BK infusion (n=6). Finally, inhibition of both COX isoforms with meclofenamate, in dogs treated with L-NAME (n=6), completely prevented the vasodilator and excretory actions of BK. The results of this study suggest that (1) NO and prostanoids dependent on COX-2 seem to be involved in the renal vasodilatation induced by BK, and (2) there is an interaction between NO and COX-1–derived metabolites in mediating the natriuretic and diuretic response to BK.

The aim of this study was to examine the relative contribution of both cyclooxygenase (COX) isoforms in producing the prostaglandins (PG) involved in the regulation of renal function, when nitric oxide (NO) synthesis is reduced. In anesthetized dogs with reduction of NO synthesis, the renal effects of a nonisozyme-specific COX inhibitor (meclofenamate) were compared with those elicited by a selective COX-2 inhibitor (nimesulide) before and during an extracellular volume expansion (ECVE). Intrarenal N G- nitro-l-arginine methyl ester (l-NAME) infusion (1 μg · kg−1 · min−1; n = 6) did not elicit renal hemodynamic changes and reduced ( P < 0.01) the renal excretory response to ECVE. Intravenous nimesulide (5 μg · kg−1 · min−1; n = 6) did not modify renal hemodynamic and reduced ( P < 0.05) sodium excretion before ECVE. Simultaneousl-NAME and nimesulide infusion ( n = 7) elicited an increment (37%) in renal vascular resistance (RVR; P < 0.05) before ECVE and no hemodynamic changes during ECVE. The reduced excretory response elicited byl-NAME and nimesulide was similar to that found duringl-NAME infusion. Finally, simultaneous l-NAME and meclofenamate infusion (10 μg · kg−1 · min−1; n = 7) induced an increase in RVR (91%, P < 0.05), a decrease in glomerular filtration rate (35%, P < 0.05), and a reduction of the renal excretory response to ECVE that was greater ( P < 0.05) than that elicited by l-NAME alone. The results obtained support the notion that PG involved in regulating renal hemodynamic and excretory function when NO synthesis is reduced are mainly dependent on COX-1 activity.

We examined the renal functional and hemodynamic changes induced by prolonged cyclooxygenase (COX) inhibition when angiotensin II levels are elevated during several consecutive days. The effects induced by the infusion of either initially subpressor or pressor angiotensin II doses (1 and 5 ng/kg/min) were examined in dogs with or without the simultaneous infusion of meclofenamate (5 microg/kg/min). Experiments were performed in conscious permanently instrumented dogs. Infusion of the lower angiotensin II dose alone (n = 6) caused a late 12+/-2% increase in arterial pressure, a 25+/-6% decrease in renal blood flow (RBF), and a transitory decrease in urinary sodium excretion. COX inhibition reduced the hypertension and renal vasoconstriction, but enhanced the sodium retention, induced by the lower dose angiotensin II infusion (n = 6). The higher angiotensin II dose (n = 6) caused a 25+/-4% increase in arterial pressure, a 24+/-5% decrease in RBF, and a transitory decrease in urinary sodium excretion. Finally, COX inhibition did not modify the renal effects elicited by the higher angiotensin II dose (n = 6). The results of this study suggest that endogenous prostaglandins play an important role in the regulation of the renal and systemic changes induced by prolonged administration of initially subpressor angiotensin II doses. It has also been demonstrated that prolonged COX inhibition does not modify the renal functional and hemodynamic changes elicited by the long-term infusion of a pressor angiotensin II dose.

In this issue of Blood, Jitschin et al demonstrate a microenvironmental glycolytic shift in chronic lymphocytic leukemia (CLL) cells mediated by Notch-c-Myc signaling. Interfering in the Notch-c-Myc pathway and reprogramming glycolytic metabolism could contribute to overcoming drug resistance in CLL.

Objectives To examine the role of nitric oxide in the cardiovascular system in spontaneous hypertension. In particular, we wanted to know whether the production of nitric oxide in the cardiovascular system of the spontaneously hypertensive rat is different from that of the normotensive Wistar-Kyoto rat and whether nitric oxide is biologically effective in this system. Design We studied various aspects of the L-arginine-nitric oxide pathway in the cardiovascular system of spontaneously hypertensive rats and Wistar-Kyoto rats. Methods To address the first objective we analysed the expression of endothelial nitric oxide synthase in the heart by Western blotting and the activity of constitutive nitric oxide synthase in resistance microvessels obtained from the mesenterium, both from spontaneously hypertensive rats and Wistar-Kyoto rats aged 14-18 weeks. We also analysed the concentration of the oxidative product of nitric oxide, nitrate, in plasma from these rats. To address the second objective, that is, to assess the bioactivity of nitric oxide, we studied the accumulation in tissue of cyclic guanosine 3',5'-monophosphate (GMP), as well as the acute and chronic effects of withdrawing the nitric oxide vasodilatory tone with the inhibitor of nitric oxide synthesis N G -nitro-L-arginine methyl ester on Wistar-Kyoto rats and spontaneously hypertensive rats. Results We found that the expression of endothelial nitric oxide synthase in the heart, the activity of constitutive nitric oxide synthase in resistance microvessels and the concentration of nitrate in plasma were all significantly higher in the spontaneously hypertensive rats. In contrast, neither cyclic GMP levels nor the effects of N G -nitro-L-arginine methyl ester were greater in the spontaneously hypertensive rat than they were in the Wistar-Kyoto rat Conclusions The nitric oxide pathway is upregulated in the resistance circulation and the heart of the spontaneously hypertensive rat by a mechanism involving induction of the constitutive nitric oxide synthase and overproduction of nitric oxide. However, nitric oxide is not sufficiently bioactive to stimulate the formation of cyclic GMP and to maintain an adequate nitric oxide-dependent vasodilatory tone.

A 3.7-Mb region of rat chromosome 13 (45.2–49.0 Mb) affects blood pressure (BP) in females only, indicating the presence of sex-specific BP loci in close proximity to the Renin locus. In the present study, we used a series of Dahl salt-sensitive/Mcwi-13 Brown Norway congenic rat strains to further resolve BP loci within this region. We identified 3 BP loci affecting female rats only, of which the 2 smaller loci (line9 BP3 and line9 BP4 ) were functionally characterized by sequence and expression analysis. Compared with SS (SS/HsdMcwiCrl), the presence of a 591-kb region of BN (BN/NHsdMcwi) chromosome 13 (line9 BP3 ) significantly lowered BP by 21 mm Hg on an 8% NaCl diet (153±7 versus 174±5 mm Hg; P BP4 ) completely erased the female-specific BP protection on 8% NaCl diet, suggesting that BN hypertensive allele(s) reside in this region. The congenic interval of the protective line 9F strain contains 3 genes ( Optc , Prelp , and Fmod ), and the hypertensive line 9E contains 1 additional gene ( Btg2 ). Sequence analysis of the 2 BP loci revealed a total of 282 intergenic variants, with no coding variants. Analysis of gene expression by quantitative real-time polymerase chain reaction revealed strain- and sex-specific differences in Prelp , Fmod , and Btg2 expression, implicating these as novel candidate genes for female-specific hypertension.

Many lines of evidence demonstrate that genetic variability contributes to chronic kidney disease susceptibility in humans as well as rodent models. Little progress has been made in discovering causal kidney disease genes in humans mainly due to genetic complexity. Here, we use a minimal congenic mapping strategy in the FHH (fawn hooded hypertensive) rat to identify Sorcs1 as a novel renal disease candidate gene. We investigated the hypothesis that genetic variation in Sorcs1 influences renal disease susceptibility in both rat and human. Sorcs1 is expressed in the kidney, and knocking out this gene in a rat strain with a sensitized genome background produced increased proteinuria. In vitro knockdown of Sorcs1 in proximal tubule cells impaired protein trafficking, suggesting a mechanism for the observed proteinuria in the FHH rat. Since Sorcs1 influences renal function in the rat, we went on to test this gene in humans. We identified associations between single nucleotide polymorphisms in SORCS1 and renal function in large cohorts of European and African ancestry. The experimental data from the rat combined with association results from different ethnic groups indicates a role for SORCS1 in maintaining proper renal function.

Chronic lymphocytic leukemia (CLL) is frequently associated with autoimmune hemolytic anemia (AIHA). However, the mechanisms governing the association between CLL and AIHA are poorly understood. MicroRNAs (miRNAs) have been associated with different clinico-biological forms of CLL and are also known to play a substantial role in autoimmunity. However, there are no studies correlating miRNA expression with the likelihood that patients with CLL will develop AIHA. In this study, we found that malignant B-cells from patients with CLL subsequently developing AIHA present nine down-regulated (i.e. miR-19a, miR-20a, miR-29c, miR-146b-5p, miR-186, miR-223, miR-324-3p, miR-484 and miR-660) miRNAs. Interestingly, two of these miRNAs (i.e. miR-20a and miR-146b-5p) are involved in autoimmune phenomena, and one (i.e. miR-146b-5p) in both autoimmunity and CLL. Furthermore, we demonstrated that miR-146b-5p modulates CD80, a molecule associated with the B-T-cell synapse and in restoration of the antigen presenting cell capacity of CLL cells.

Studies of transcriptome profiles have provided new insights into mechanisms underlying the development of hypertension. Cell type heterogeneity in tissue samples, however, has been a significant hindrance in these studies. We performed a transcriptome analysis in medullary thick ascending limbs of the loop of Henle isolated from Dahl salt-sensitive rats. Genes differentially expressed between Dahl salt-sensitive rats and salt-insensitive consomic SS.13 BN rats on either 0.4% or 7 days of 8.0% NaCl diet (n=4) were highly enriched for genes located on chromosome 13, the chromosome substituted in the SS.13 BN rat. A pathway involving cell proliferation and cell cycle regulation was identified as one of the most highly ranked pathways based on differentially expressed genes and by a Bayesian model analysis. Immunofluorescent analysis indicated that just 1 week of a high-salt diet resulted in a severalfold increase in proliferative medullary thick ascending limb cells in both rat strains, and that Dahl salt-sensitive rats exhibited a significantly greater proportion of medullary thick ascending limb cells in a proliferative state than in SS.13 BN rats (15.0±1.4% versus 10.1±0.6%; n=7–9; P

the physiological genomics (PG) Group of the American Physiological Society marked the 125th anniversary celebration of the American Physiological Society by organizing four exciting scientific sessions at the recently concluded Experimental Biology meeting 2012 held at the San Diego Convention Center. These included a cross-sectional workshop “Tool-kit for genomic biomarker discovery by physiologists” chaired by Drs. Bina Joe and Lance Miller, a symposium on “Molecular and cellular therapy for cardiovascular disease” chaired by Drs. Zhongjie Sun and Stephen C. Mockrin, a featured topic on “Translational biomarkers of hypertension: Insights from animal models” chaired by Dr. Joe, and a special session, “Trainee-highlights in physiological genomics” chaired by Drs. Jia Zhuo and Carol Moreno. The scientific impact of these four sessions was reflected through the active discussions that followed after each of these session presentations and from large scientific audiences including faculty members and postdoctoral and graduate trainees across all sections of the American Physiological Society.

BACKGROUND The Fcγ receptor IIb (FcγRIIb) is an inhibitory Fcγ receptor that suppresses B-cell activation when coligated with B-cell antigen receptor (BCR). Previous studies from our group indicate that the ability of the FcγRIIb to inhibit BCR signaling after coligation is attenuated in Chronic Lymphocytic Leukemia (CLL). Furthermore, in contrast to what has been described in normal murine B-cells, stimulation of the FcγRIIb alone induces proliferation of CLL cells. However, the correlation between FcγRIIb expression, immunophenotypic characteristics, and clinical variables in patients with CLL has not been studied. AIM The aim of this study was to correlate the expression of FcγRIIb on leukemic cells from previously untreated CLL patients and its immunophenotypic features and clinical parameters. METHODS The study population included 112 patients with untreated CLL for whom cryopreserved peripheral blood samples were available before treatment. The diagnosis was based on IWCLL 2008 criteria. The median patients' follow up was 57.92 months (range: 2.23-439.78 months). FcγRIIb expression levels were determined by flow cytometry on CD5+/CD19+ CLL cells using a specific Alexa488-conjugated murine mAb specific for human FcγRIIb. The following combinations were assessed: FcγRIIb/CD38/CD19/CD5, FcγRIIb/CD49d/CD19/CD5, and FcγRIIb/CD69/CD19/CD5. Results were expressed as the ratio between the MFI for FcγRIIb and the MFI for the corresponding isotype (MFIR). FcγRIIb expression levels were correlated with: i) expression of CD49d, CD38 and CD69, ii) clinico-biological characteristics, and iii) clinical outcome. Differences of FcγRIIb expression on dichotomized clinicopathological variables were assessed with Mann Whitney test. Kaplan-Meier survival and Cox regression analysis were performed to evaluate the correlation of FcγRIIb expression with clinical outcome. Best cut-offs for overall survival (OS) and treatment-free survival (TFS) were determined by ROC curves. RESULTS All CD5+CD19+ leukemic cells samples expressed FcγRIIb. However, FcγRIIb expression levels markedly varied between patients (median MFIR: 45.8; interquartile range: 14.9-76.6; 5th -95th percentile: 17.15-111.4). FcγRIIb expression was significantly higher in patients who had high (≥30%) CD49d expression than in those with low ( In univariate analysis, low FcγRIIb expression levels (MFIR< 26.67) were associated with shorter OS (HR 4.01, 95%CI 1.15-13.90, p=0.029), together with older age, advanced stage, and expression of CD38 and CD49d. Advanced stage, unmutated IGHV, and CD38, CD49d and ZAP-70 expression were also associated significantly with shorter TFS. Thus, patients with higher levels of FcγRIIb had better survival than those with lower levels (Log rank test, p = 0.018). A multivariate analysis adjusted for FcγRIIb expression, age, disease stage, CD38, and CD49d identified older age (≥65 yrs) (HR 150.76, 95%CI 5.39-4212.42, p =0.003), low FcγRIIb expression (HR 111.91, 95%CI 6.71-1866.97, p =0.001), advanced stage (B/C) (HR 17.44, 95%CI 1.45-210.24, p =0.024) and CD38 expression (HR 5.02, 95%CI 1.01-25.18, p =0.050) as independent predictors for shorter OS. CONCLUSIONS In this study, FcγRIIb expression on leukemic cells from untreated patients with CLL was found to be an independent prognostic marker for OS, overcoming the prognostic value of CD49d, which is consistent with the key role of the FcγRIIb in the pathogenesis of CLL. Further analysis aimed at validating this observation and to better understand the functional cooperation of FcγRIIb with other molecules, particularly CD49d, are warranted. These studies could open a new venue in CLL treatment. Disclosures Gorlatov: MacroGenics: Employment. Sierra:Novartis: Research Funding; Celgene: Research Funding; Amgen: Research Funding.

Transfer of chromosome 13 from the Brown Norway (BN) rat onto the Dahl salt-sensitive (SS) genetic background attenuates the development of hypertension, but the genes involved remain to be identified. The purpose of the present study was to confirm by telemetry that a congenic strain [SS.BN-(D13Hmgc37-D13Got22)/Mcwi, line 5], carrying a 13.4-Mb segment of BN chromosome 13 from position 32.4 to 45.8 Mb, is protected from the development of hypertension and then to narrow the region of interest by creating and phenotyping 11 additional subcongenic strains. Mean arterial pressure (MAP) rose from 118 ± 1 to 186 ± 5 mmHg in SS rats fed a high-salt diet (8.0% NaCl) for 3 wk. Protein excretion increased from 56 ± 11 to 365 ± 37 mg/day. In contrast, MAP only increased to 152 ± 9 mmHg in the line 5 congenic strain. Six subcongenic strains carrying segments of BN chromosome 13 from 32.4 and 38.2 Mb and from 39.9 to 45.8 Mb were not protected from the development of hypertension. In contrast, MAP was reduced by ∼30 mmHg in five strains, carrying a 1.9-Mb common segment of BN chromosome 13 from 38.5 to 40.4 Mb. Proteinuria was reduced by ∼50% in these strains. Sequencing studies did not identify any nonsynonymous single nucleotide polymorphisms in the coding region of the genes in this region. RT-PCR studies indicated that 4 of the 13 genes in this region were differentially expressed in the kidney of two subcongenic strains that were partially protected from hypertension vs. those that were not. These results narrow the region of interest on chromosome 13 from 13.4 Mb (159 genes) to a 1.9-Mb segment containing only 13 genes, of which 4 are differentially expressed in strains partially protected from the development of hypertension.

Rf-3, a quantitative trait locus (QTL) on rat chromosome 3, affects the development of CKD in Fawn-Hooded Hypertensive (FHH) rats. This QTL spans 110 Mb and approximately 1400 genes; therefore, narrowing the position of this locus is necessary to elucidate potential candidate genes. Here, we used congenic models and comparative genomics to refine the Rf-3 candidate region. We generated congenic lines carrying smaller intervals (subcongenics) of the Rf-3 region and used these lines to reduce the Rf-3 candidate region by 94% (to 7.1 Mb). We used comparative genomics to identify QTL for both nephropathy and albuminuria in the syntenic region of this interval for both human and mouse. We also used the overlapping homologous regions to reduce the number of likely positional candidate genes to 13 known or predicted genes. By combining congenic models and cross-species studies, we narrowed the list of candidate genes to a level that we could sequence the whole interval to further identify the causative gene in future studies.

We here report the genetic basis for susceptibility and resistance to carcinogen-mediated [7,12-dimethylbenz[a]anthracene (DMBA)] mammary tumorigenesis using the full panel of SS/BN consomic rat strains, in which substitutions of individual chromosomes from the resistant BN strain onto the genomic background of the susceptible SS strain were made. Analysis of 252 consomic females identified rat mammary Quantitative Trait Loci (QTLs) affecting tumor incidence on chromosomes 3 and 5, latency on chromosomes 3, 9, 14, and 19, and multiplicity on chromosomes 13, 16, and 19. In addition, we unexpectedly identified a novel QTL on chromosome 6 controlling a lethal toxic phenotype in response to DMBA. Upon further investigation with chromosomes 6 and 13 congenic lines, in which an additional 114 rats were investigated, we mapped (1) a novel mammary tumor QTL to a region of 27.1 Mbp in the distal part of RNO6, a region that is entirely separated from the toxicity phenotype, and (2) a novel and powerful mammary tumor susceptibility locus of 4.5 Mbp that mapped to the proximal q-arm of RNO13. Comparison of genetic strain differences using existing rat genome databases enabled us to further construct priority lists containing single breast cancer candidate genes within the defined QTLs, serving as potential functional variants for future testing.