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Chronic lymphocytic leukemia (CLL) is frequently associated with autoimmune hemolytic anemia and immune thrombocytopenia and, less frequently, with pure red cell aplasia and immune neutropenia. The emergence of these complications is related to an intertwined and complex relationship between patient, disease, and treatment characteristics. The prognostic repercussion of autoimmune cytopenia (AIC) in patients with CLL mainly depends on its response to therapy. For patients with AIC and nonactive CLL, treatment is as in primary, uncomplicated AIC, keeping in mind that no response is an indication for CLL therapy. The success of treating active CLL-related AIC widely relies on a flexible strategy that should include initial therapy with corticosteroids and a rapid shift to effective CLL therapy in nonresponding patients. Targeted therapies (e.g., ibrutinib) that have already demonstrated to be effective in CLL-related AIC will likely offer a unique possibility of treating both AIC and CLL as a single target.

Background— Genome-wide association studies are powerful tools for nominating pathogenic variants, but offer little insight as to how candidate genes affect disease outcome. Such is the case for SH2B adaptor protein 3 ( SH2B3 ), which is a negative regulator of multiple cytokine signaling pathways and is associated with increased risk of myocardial infarction (MI), but its role in post-MI inflammation and fibrosis is completely unknown. Methods and Results— Using an experimental model of MI (left anterior descending artery occlusion/reperfusion injury) in wild-type and Sh2b3 knockout rats (Sh2b3 em2Mcwi ), we assessed the role of Sh2b3 in post-MI fibrosis, leukocyte infiltration, angiogenesis, left ventricle contractility, and inflammatory gene expression. Compared with wild-type, Sh2b3 em2Mcwi rats had significantly increased fibrosis (2.2-fold; P 2-fold; P P em2Mcwi rats (1.7-fold; P em2Mcwi rats. In total, 12 genes were significantly elevated in the post left anterior descending artery occluded/reperfused hearts of Sh2b3 em2Mcwi rats relative to wild-type, of which 3 ( NLRP12 , CCR2 , and IFNγ ) were significantly elevated in the left ventricle of heart failure patients carrying the MI-associated rs3184504 [T] SH2B3 risk allele. Conclusions— These data demonstrate for the first time that SH2B3 is a crucial mediator of post-MI inflammation and fibrosis.

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.

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

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.

To analyze recent scientific contributions correlating genetic lesions with clinical features in chronic lymphocytic leukemia (CLL).Genetic abnormalities are increasingly important to understand not only CLL biology but also other aspects such as its epidemiology, diagnosis, prognosis, and clinical evolution.Genetic abnormalities provide important clues in understanding the clinical and biologic heterogeneity of CLL, assessing the individual risk of each patient with this form of leukemia and also predicting response to therapy.

Background: Genome wide association studies (GWAS) are powerful tools for nominating pathogenic variants, but offer little insight as to how candidate genes impact disease outcome. Such is the case for SH2B adaptor protein 3 (SH2B3), which is associated with coronary artery disease (CAD), atherosclerosis, and risk of myocardial infarction (MI), but its role in post-MI response is completely unknown. Methods: Using an experimental model of MI (left anterior descending artery [LAD] occlusion) in wild-type (WT) and Sh2b3 knockout (KO) rats, we assessed the role of Sh2b3 in post-MI fibrosis, leukocyte infiltration, angiogenesis, left ventricle (LV) contractility, and inflammatory gene expression. We also confirmed our findings in LV samples from end-stage heart failure patients with or without the MI-associated SH2B3 risk allele. Results: Compared with WT, Sh2b3 KO rats had significantly increased fibrosis (2.2-fold; P2-fold; P Conclusions: These data suggest for the first time that SH2B3 is a master risk factor for MI by impacting both MI incidence and post-MI response.

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.

We have shown that NO production, assessed by measuring changes in plasma nitrate concentration, is down-regulated when blood pressure falls. This study intended to determine first, whether NO-derived plasma nitrate varies in response to increases in blood pressure induced by different mechanical and pharmacologic stimuli, including angiotensin II and catecholamines; and second, specifically to study the interaction between angiotensin II and NO production. An intravenous infusion (4-10 min) of norepinephrine (7.5 microg/kg/min), phenylephrine (30 microg/kg/min), or angiotensin II (0.3 and 3 microg/kg/min) caused hypertension accompanied by an increase in plasma nitrate, as assessed by high-performance capillary electrophoresis. Mechanical hypertension elicited by aortic occlusion also was accompanied by an increase in plasma nitrate. Angiotensin II (0.03, 0.3, and 3 microg/kg/min, 10 min) dose-dependently increased blood pressure. The intermediate and high dose, but not the low dose, of angiotensin II increased plasma nitrate concentration. N(G)-nitro-L-arginine methyl ester (L-NAME) lowered the basal concentration of plasma nitrate, abolished the increase in plasma nitrate elicited by angiotensin II and norepinephrine, and potentiated the pressor effect of the low dose of angiotensin II, although this dose did not increase NO production. L-NAME also potentiated the pressor effects of the intermediate dose of angiotensin II. This study demonstrates that an augmented systemic production of NO, measured as an increase in plasma nitrate, takes place after acute hypertension. The results of this study suggest that an increase in NO generation occurs when angiotensin II hypertension exceeds a certain limit, below which the basal production of NO is sufficient to compensate the vasoconstriction.

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.

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.

Background: Genome-wide association studies (GWAS) are frequently used to nominate candidate genes for complex diseases, but are largely unable to identify the causative variant(s). One example: the AGTRAP-PLOD1 locus contains 6 genes in close proximity that are all associated with blood pressure (BP), making it difficult to delineate specific allele(s) that underlie human hypertension at this locus. Here we present a novel rat model that was developed to rapidly dissect GWAS loci using high-throughput gene mutation on a single hypertensive background. METHODS: Agtrap, Mthfr, Clcn6, Nppa, Nppb, and Plod1 were individually mutated in the Dahl SS rat by zinc finger nuclease (ZFN) injections. ZFN-mutated and wild type (WT) control SS rats (n=8-25 per group) were assessed for BP and renal damage after 10 days on a 4% NaCl diet. Temporal gene expression during development of hypertension was assessed by qRT-PCR and confirmed by RNA-seq. Results: Expression of all AGTRAP-PLOD1 transcripts changed significantly in response to elevated BP, indicating possible roles of the entire AGTRAP-PLOD1 locus in salt-sensitive hypertension. However, compared with WT, only NPPA mutation further increased mean BP (Δ+27mmHg, P Conclusions: Combined with human GWAS, our data show for the first time that NPPA and CLCN6 are divergent mediators of BP at the AGTRAP-PLOD1 locus, while MTHFR mutation directly increases susceptibility to end-stage renal disease. These novel mechanistic data provide rationale for developing haplotype-specific therapies for treating hypertension.

Whole and partial chromosome introgression from the Brown Norway (BN) rat into the Dahl Salt Sensitive (SS) rat is a valuable tool for identifying genes and pathways associated with disease phenotypes in the SS rat. We have created two congenic lines with BN substitutions differing by 22.9 Kbp. The congenic regions differ by one gene, Btg2 , and have 35 sequence variants, three of which occur in Btg2 . We have characterized angiogenesis and development of hypertension in these new strains, and since Btg2 is known to be modulated by estradiol, we measured both phenotypes in males and females. Mean arterial pressure (MAP) was measured by telemetry after three weeks of 8% NaCl diet. Angiogenesis was measured by electrical stimulation of one hindlimb (Tibialis anterior, TA, and Extensor digitorum longus, EDL) with the contralateral leg acting as control. Renal damage was assessed by 24-h protein/albumin excretion after two weeks on 8% NaCl diet. Btg2 expression was determined by QRT-PCR on renal cortex and medulla as well as stimulated and unstimulated TA muscle. Males from both lines had the same MAP following three weeks of 8% NaCl diet (Btg2 BN : 187±5 mmHg, Btg2 SS : 185±3). Females with Btg2 BN had significantly higher MAP (180±6 mmHg) than Btg2 SS females (153±7 mmHg). Angiogenesis in response to electrical stimulation was observed in Btg2 BN males (TA=20.0±4.5% increase in vessel density, EDL=15.8±5.8%), but not in Btg2 SS males (TA= 5.1±2.2%, EDL=4.4±2.7%). Females of both strains had angiogenesis (Btg2 BN : TA=9.8±6%, EDL=8.5±1.2%; Btg2 SS : TA=12.7±2.4%, EDL=12.1±4.1%). Btg2 BN females had significantly higher protein excretion than SS and Btg2 SS was significantly lower; males had no significant differences. Btg2 mRNA expression in male skeletal muscle displayed a 2-fold increase (stimulated to unstimulated) in Btg2 BN and a 3.5-fold increase in Btg2 SS . Males had no differences in expression in renal cortex or medulla, but females had significant differences in cortex (Btg2 BN : 1.8±0.2 fold change, Btg2 SS : 0.4±.01). These data suggest Btg2 expression may contribute to the development of hypertension and inhibited angiogenic response in SS rats, with gender-specific differences in the expression of this gene.

The data revealed by GWAS studies provide a wealth of candidate human disease genes now requiring functional validation in animal models. In two years, we disrupted a large set of GWAS candidate genes for human hypertension and chronic kidney disease by Zinc Finger Nuclease (ZFN)-mediated gene targeting in the SS (Dahl salt-sensitive) strain background. Phenotyping male rats from gene-disrupted strains revealed five genes ( Sh2b3 , Rasgrp3 , Gpr73 , Ulk3 , and Wdr72 ) significantly altering the salt-induced hypertension in and renal damage phenotypes in this model strain of human hypertension compared to age matched wild type controls. Ingenuity Pathway Analysis revealed a putative connection for four of these genes ( Sh2b3 , Rasgrp3 , Gpr73 , and Ulk3 ) to the ERK/MAPK signaling pathway and suggested that decreased ERK signaling would exacerbate disease in the SS model. To test this hypothesis, we measured the response of SS rats to a 4% salt diet with daily IP injections of the MEK inhibitor PD98059 or vehicle control. Wild type SS animals treated with PD98059 show a significant increase in mean arterial pressure compared to vehicle-injected controls (n=10, 142±7 vs. 126±6 mmHg respectively, P participation of multiple genes in the ERK/MAPK signaling pathway indicates that regulation of this pathway plays a major role in determining blood pressure.