Positional cloning of quantitative trait nucleotides for blood pressure and cardiac QT-interval by targeted CRISPR/Cas9 editing of a novel long non-coding RNA.

Multiple GWAS studies have reported strong association of cardiac QT-interval to a region on HSA17. Interestingly, a rat locus homologous to this region is also linked to QT-intervals. The high resolution positional mapping study located the rat QT-interval locus to a <42.5kb region on RNO10. This region contained no variants in protein-coding sequences, but a prominent contiguous 19bp indel polymorphism was noted within a novel predicted long non-coding RNA (lncRNA), which we named as Rffl-lnc1. To assess the candidacy of this novel lncRNA on QT-interval, targeted CRISPR/Cas9 based genome-engineering approaches were applied on the rat strains used to map this locus. Targeted disruption of the rat Rffl-lnc1 locus caused aberrant, short QT-intervals and elevated blood pressure. Further, to specifically examine the significance of the 19bp polymorphism within the Rffl-lnc1 locus, a CRISPR/Cas9 based targeted knock-in rescue model was constructed by inserting the 19bp into the strain which contained the deletion polymorphism. The knock-in alleles successfully rescued the aberrant QT-interval and blood pressure phenotypes. Further studies revealed that the 19bp polymorphism was necessary and sufficient to recapitulate the phenotypic effect of the previously mapped <42.5kb rat locus. To our knowledge, this study is the first demonstration of a combination of both CRISPR/Cas9 based targeted disruption as well as CRISPR/Cas9 based targeted knock-in rescue approaches applied for a mammalian positional cloning study, which defines the quantitative trait nucleotides (QTNs) within a rat long non-coding RNA as being important for the pleiotropic regulation of both cardiac QT-intervals and blood pressure.

Targeted disruption of regulated endocrine-specific protein ( Resp18) in Dahl SS/Mcw rats aggravates salt-induced hypertension and renal injury.

Hypertension is a classic example of a complex polygenic trait, impacted by quantitative trait loci (QTL) containing candidate genes thought to be responsible for blood pressure (BP) control in mammals. One such mapped locus is on rat chromosome 9, wherein the proof for a positional candidate gene, regulated endocrine-specific protein-18 ( Resp18) is currently inadequate. To ascertain the status of Resp18 as a BP QTL, a custom targeted gene disruption model of Resp18 was developed on the Dahl salt-sensitive (SS) background. As a result of this zinc-finger nuclease (ZFN)-mediated disruption, a 7 bp deletion occurred within exon 3 of the Resp18 locus. Targeted disruption of Resp18 gene locus in SS rats decreases its gene expression in both heart and kidney tissues regardless of their dietary salt level. Under a high-salt dietary regimen, both systolic and diastolic BP of Resp18mutant rats were significantly increased compared with SS rats. Resp18mutant rats demonstrated increased renal damage, as evidenced by higher proteinuria and increased renal fibrosis compared with SS rats. Furthermore, under a high-salt diet regimen, the mean survival time of Resp18mutant rats was significantly reduced compared with SS rats. These findings serve as evidence in support of Resp18 as a gene associated with the development of hypertension and renal disease.

Targeted disruption of Cd40 in a genetically hypertensive rat model attenuates renal fibrosis and proteinuria, independent of blood pressure.

High blood pressure is a common cause of chronic kidney disease. Because CD40, a member of the tumor necrosis factor receptor family, has been linked to the progression of kidney disease in ischemic nephropathy, we studied the role of Cd40 in the development of hypertensive renal disease. The Cd40 gene was mutated in the Dahl S genetically hypertensive rat with renal disease by targeted-gene disruption using zinc-finger nuclease technology. These rats were then given low (0.3%) and high (2%) salt diets and compared. The resultant Cd40 mutants had significantly reduced levels of both urinary protein excretion (41.8 ± 3.1 mg/24 h vs. 103.7 ± 4.3 mg/24 h) and plasma creatinine (0.36 ± 0.05 mg/dl vs. 1.15 ± 0.19 mg/dl), with significantly higher creatinine clearance compared with the control S rats (3.04 ± 0.48 ml/min vs. 0.93 ± 0.15 ml/min), indicating renoprotection was conferred by mutation of the Cd40 locus. Furthermore, the Cd40 mutants had a significant attenuation in renal fibrosis, which persisted on the high salt diet. However, there was no difference in systolic blood pressure between the control and Cd40 mutant rats. Thus, these data serve as the first evidence for a direct link between Cd40 and hypertensive nephropathy. Hence, renal fibrosis is one of the underlying mechanisms by which Cd40 plays a crucial role in the development of hypertensive renal disease.

High-resolution mapping of a novel rat blood pressure locus on chromosome 9 to a region containing the Spp2 gene and colocalization of a QTL for bone mass.

Through linkage analysis of the Dahl salt-sensitive (S) rat and the spontaneously hypertensive rat (SHR), a blood pressure (BP) quantitative trait locus (QTL) was previously located on rat chromosome 9. Subsequent substitution mapping studies of this QTL revealed multiple BP QTLs within the originally identified logarithm of odds plot by linkage analysis. The focus of this study was on a 14.39 Mb region, the distal portion of which remained unmapped in our previous studies. High-resolution substitution mapping for a BP QTL in the setting of a high-salt diet indicated that an SHR-derived congenic segment of 787.9 kb containing the gene secreted phosphoprotein-2 (Spp2) lowered BP and urinary protein excretion. A nonsynonymous G/T polymorphism in the Spp2 gene was detected between the S and S.SHR congenic rats. A survey of 45 strains showed that the T allele was rare, being detected only in some substrains of SHR and WKY. Protein modeling prediction through SWISSPROT indicated that the predicted protein product of this variant was significantly altered. Importantly, in addition to improved cardiovascular and renal function, high salt-fed congenic animals carrying the SHR T variant of Spp2 had significantly lower bone mass and altered bone microarchitecture. Total bone volume and volume of trabecular bone, cortical thickness, and degree of mineralization of cortical bone were all significantly reduced in congenic rats. Our study points to opposing effects of a congenic segment containing the prioritized candidate gene Spp2 on BP and bone mass.

Evidence for a link between gut microbiota and hypertension in the Dahl rat.

The gut microbiota plays a critical role in maintaining physiological homeostasis. This study was designed to evaluate whether gut microbial composition affects hypertension. 16S rRNA genes obtained from cecal samples of Dahl salt-sensitive (S) and Dahl salt-resistant (R) rats were sequenced. Bacteria of the phylum Bacteroidetes were higher in the S rats compared with the R rats. Furthermore, the family S24-7 of the phylum Bacteroidetes and the family Veillonellaceae of the phylum Firmicutes were higher in the S rats compared with the R rats. Analyses of the various phylogenetic groups of cecal microbiota revealed significant differences between S and R rats. Both strains were maintained on a high-salt diet, administered antibiotics for ablation of microbiota, transplanted with S or R rat cecal contents, and monitored for blood pressure (BP). Systolic BP of the R rats remained unaltered irrespective of S or R rat cecal transplantation. Surprisingly, compared with the S rats given S rat cecal content, systolic BP of the S rats given a single bolus of cecal content from R rats was consistently and significantly elevated during the rest of their life, and they had a shorter lifespan. A lower level of fecal bacteria of the family Veillonellaceae and increased plasma acetate and heptanoate were features associated with the increased BP observed in the S rats given R rat microbiota compared with the S rats given S rat microbiota. These data demonstrate a link between microbial content and BP regulation and, because the S and R rats differ in their genomic composition, provide the necessary basis to further examine the relationship between the host genome and microbiome in the context of BP regulation in the Dahl rats.

Isolation and high-throughput sequencing of two closely linked epistatic hypertension susceptibility loci with a panel of bicongenic strains.

Interactions or epistasis between genetic factors may contribute to "missing heritability." While linkage analyses detect epistasis, defining the limits of the interacting segments poses a significant challenge especially when the interactions are between loci in close proximity. The goal of the present study was to isolate two such epistatic blood pressure (BP) loci on rat chromosome 5. A panel of S.LEW bicongenic strains along with the corresponding monocongenic strains was constructed. BP of each set comprising of one bicongenic and two corresponding monocongenic strains were determined along with the parental Salt-sensitive (S) strain. Epistasis was observed in one out of four sets of congenic strains, wherein systolic blood pressures (SBP) of the two monocongenic strains S.LEW(5)x6Bx9x5a and S.LEW(5)x6Bx9x5b were comparable to that of S, but the SBP of the bicongenic strain S.LEW(5)x6Bx9x5 (157 ± 4.3 mmHg) was significantly lower than that of S (196 ± 6.8 mmHg, P < 0.001). A two-way ANOVA indicated significant interactions between the LEW alleles at the two loci. The interacting loci were 2.02 Mb apart and located within genomic segments spanning 7.77 and 4.18 Mb containing 7,360 and 2,753 candidate variants, respectively. The current study demonstrates definitive evidence for epistasis and provides genetic tools for further dissection of the isolated epistatic BP loci.

Intrarenal Dopaminergic System Is Dysregulated in SS-Resp18mutant Rats.

The genetic and molecular basis of developing high blood pressure and renal disease are not well known. Resp18mutant Dahl salt-sensitive (SS-Resp18mutant) rats fed a 2% NaCl diet for six weeks have high blood pressure, increased renal fibrosis, and decreased mean survival time. Impairment of the dopaminergic system also leads to hypertension that involves renal and non-renal mechanisms. Deletion of any of the five dopamine receptors may lead to salt-sensitive hypertension. Therefore, we investigated the interaction between Resp18 and renal dopamine in SS-Resp18mutant and Dahl salt-sensitive (SS) rats. We found that SS-Resp18mutant rats had vascular dysfunction, as evidenced by a decrease in vasorelaxation in response to sodium nitroprusside. The pressure-natriuresis curve in SS-Resp18mutant rats was shifted down and to the right of SS rats. SS-Resp18mutant rats had decreased glomerular filtration rate and dopamine receptor subtypes, D1R and D5R. Renal dopamine levels were decreased, but urinary dopamine levels were increased, which may be the consequence of increased renal dopamine production, followed by secretion into the tubular lumen. The increased renal dopamine production in SS-Resp18mutant rats in vivo was substantiated by the increased dopamine production in renal proximal tubule cells treated with L-DOPA. Overall, our study provides evidence that targeted disruption of the Resp18 locus in the SS rat dysregulates the renal dopaminergic system.

Effect of pomegranate juice on Angiotensin II-induced hypertension in diabetic Wistar rats.

Acute subcutaneous administration of Angiotensin II (Ang II) causes a rise in blood pressure in diabetic Wistar rats. Diabetes was induced using streptozotocin (70 mg/kg, i.v.). Chronic administration of pomegranate juice (PJ) extract (100 mg/kg and 300 mg/kg; p.o. for 4 weeks) obtained from Punica granatum (punicaceae) fruits reduced the mean arterial blood pressure and vascular reactivity changes to various catecholamines and also reversed the biochemical changes induced by diabetes and Ang II. PJ treatment also caused a significant decrease in levels of thiobarbituric acid reactive substances (TBARS) in kidney and pancreas while activities of enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GSH) showed significant elevation. The cumulative concentration response curve (CCRC) of Ang II was shifted towards right in rats treated with PJ using isolated strip of ascending colon. In histopathological examination, PJ treatment prevented the tubular degenerative changes induced by diabetes. The results suggest that the PJ extract could prevent the development of high blood pressure induced by Ang II in diabetic rats probably by combating the oxidative stress induced by diabetes and Ang II and by inhibiting ACE activity. In conclusion, PJ has antihypertensive action in Ang II diabetic model.

Fine mapping of epistatic genetic determinants of blood pressure on rat chromosome 5.

BACKGROUND: Previously, using linkage analysis and substitution mapping, two closely-linked interactive blood pressure quantitative trait loci (QTLs), BP QTL1 and BP QTL2, were located within a 13.96 Mb region from 117894038 to 131853815 bp (RGSC 3.4 version) on rat chromosome 5 (RNO5). This was done by using a series of congenic strains consisting of genomic segments of the Dahl salt-sensitive (S) rat substituted with that of the normotensive Lewis (LEW) rat. The interactive nature of the two loci was further confirmed by the construction and characterization of a panel of S.LEW bicongenic strains and corresponding S.LEW monocongenic strains, which provided definitive evidence of epistasis (genetic interaction) between BP QTL1 (7.77 Mb) and BP QTL2 (4.18 Mb). The purpose of this work was to further map these interacting QTLs. METHOD: A new panel of seven new S.LEW bicongenic strains was constructed and characterized for BP. RESULTS: The data obtained from these new strains further resolved BP QTL1 from 7.77 to 2.93 Mb. Further, BP QTL2 was traceable as not being a single QTL, but a composite of at least three QTLs, LEW alleles at two of which located within 2.26 Mb and 175 kb lowered BP but the third one located within 1.31 Mb increased BP. CONCLUSION: Lack of coding variation within any of the regions further mapped within the previous QTL2 suggests noncoding variation as likely responsible for the observed epistasis.

Attenuation of Microbiotal Dysbiosis and Hypertension in a CRISPR/Cas9 Gene Ablation Rat Model of GPER1.

G-protein-coupled estrogen receptor, Gper1, has been implicated in cardiovascular disease, but its mechanistic role in blood pressure control is poorly understood. Here, we demonstrate that genetically salt-sensitive hypertensive rats with complete genomic excision of Gper1 by a multiplexed guide RNA CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR associated proteins) approach present with lower blood pressure, which was accompanied by altered microbiota, different levels of circulating short chain fatty acids, and improved vascular relaxation. Microbiotal transplantation from hypertensive Gper1+/+ rats reversed the cardiovascular protective effect exerted by the genomic deletion of Gper1. Thus, this study reveals a role for Gper1 in promoting microbiotal alterations that contribute to cardiovascular pathology. However, the exact mechanism by which Gper1 regulates blood pressure is still unknown. Our results indicate that the function of Gper1 is contextually dependent on the microbiome, whereby, contemplation of using Gper1 as a target for therapy of cardiovascular disease requires caution.

Pleiotropic Effect of a High Resolution Mapped Blood Pressure QTL on Tumorigenesis.

This study is focused on a translationally significant, genome-wide-association-study (GWAS) locus for cardiovascular disease (QT-interval) on human chromosome 17. We have previously validated and high resolution mapped the homologous genomic segment of this human locus to <42.5 kb on rat chromosome 10. This <42.5 kb segment in rats regulates both QT-interval and blood pressure and contains a single protein-coding gene, rififylin (Rffl). The expression of Rffl in the hearts and kidneys is differential between Dahl S and S.LEW congenic rats, which are the strains used for mapping this locus. Our previous study points to altered rate of endocytic recycling as the underlying mechanism, through which Rffl operates to control both QT-interval and blood pressure. Interestingly, Rffl also contributes to tumorigenesis by repressing caspases and tumor suppressor genes. Moreover, the expression of Methyl-CpG Binding Domain Protein 2 (Mbd2) in the hearts and kidneys is also higher in the S.LEW congenic strain than the background (control) Dahl S strain. Mbd2 can repress methylated tumor suppressor genes. These data suggest that the S.LEW congenic strain could be more susceptible to tumorigenesis. To test this hypothesis, the S and S.LEW strains were compared for susceptibility to azoxymethane-induced colon tumors. The number of colon tumors was significantly higher in the S.LEW congenic strain compared with the S rat. Transcriptomic analysis confirmed that the chemical carcinogenesis pathway was significantly up-regulated in the congenic strain. These studies provide evidence for a GWAS-validated genomic segment on rat chromosome 10 as being important for the regulation of cardiovascular function and tumorigenesis.

Multiple blood pressure loci with opposing blood pressure effects on rat chromosome 1 in a homologous region linked to hypertension on human chromosome 15.

Genetic dissection of blood pressure (BP) quantitative trait loci (QTLs) in rats has facilitated the fine-mapping of regions linked to the inheritance of hypertension. The goal of the current study was to further fine-map one such genomic region on rat chromosome 1 (BPQTL1b1), the homologous region of which on human chromosome 15 harbors BP QTLs, as reported by four independent studies. Of the six substrains constructed and studied, the systolic BP of two of the congenic strains were significantly lower by 36 and 27 mm Hg than that of the salt-sensitive (S) rat (P < 0.0001, P = 0.0003, respectively). The congenic segments of these two strains overlapped between 135.12 and 138.78 Mb and contained eight genes and two predicted miRNAs. None of the annotations had variants within expressed sequences. These data taken together with the previous localization resolved QTL1b1 with a 70% improvement from the original 7.39 Mb to the current 2.247 Mb interval. Furthermore, the systolic BP of one of the congenic substrains was significantly higher by 20 mm Hg (P < 0.0001) than the BP of the S rat. The limits of this newly identified QTL with a BP increasing effect (QTL1b1a) were between 134.12 and 135.76 Mb, spanning 1.64 Mb, containing two protein-coding genes, Mctp2 and Rgma, and a predicted miRNA. There were four synonymous variants within Mctp2. These data provide evidence for two independent BP QTLs with opposing BP effects within the previously identified BP QTL1b1 region. Additionally, these findings illustrate the complexity underlying the genetic mechanisms of BP regulation, wherein inherited elements beyond protein-coding sequences or known regulatory regions could be operational.

Mutation within the hinge region of the transcription factor Nr2f2 attenuates salt-sensitive hypertension.

Genome-wide association studies (GWAS) have prioritized a transcription factor, nuclear receptor 2 family 2 (NR2F2), as being associated with essential hypertension in humans. Here we provide evidence that validates this association and indicates that Nr2f2 is a genetic determinant of blood pressure (BP). Using the zinc-finger nuclease technology, the generation of a targeted Nr2f2-edited rat model is reported. The resulting gene-edited rats have a 15 bp deletion in exon 2 leading to a five-amino-acid deletion in the hinge region of the mutant Nr2f2 protein. Both systolic and diastolic blood pressures of the Nr2f2(mutant) rats are significantly lower than controls. Because the hinge region of Nr2f2 is required for interaction with Friend of Gata2 (Fog2), protein-protein interaction is examined. Interaction of Nr2f2(mutant) protein with Fog2 is greater than that with the wild-type Nr2f2, indicating that the extent of interaction between these two transcription factors critically influences BP.