Caitlin C. O'Meara

Identifying multiple causative genes at a single GWAS locus

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.

A systematic analysis of neonatal mouse heart regeneration after apical resection

The finding that neonatal mice are able to regenerate myocardium after apical resection has recently been questioned. We determined if heart regeneration is influenced by the size of cardiac resection and whether surgical retraction of the ventricular apex results in an increase in cardiomyocyte cell cycle activity. We performed moderate or large apical ventricular resections on neonatal mice and quantified scar infiltration into the left ventricular wall at 21 days post-surgery. Moderately resected hearts had 15±2% of the wall infiltrated by a collagen scar; significantly greater scar infiltration (23±4%) was observed in hearts with large resections. Resected hearts had higher levels of cardiomyocyte cell cycle activity relative to sham hearts. Surgically retracting the ventricle often resulted in fibrosis and induced cardiomyocyte cell cycle activity that were comparable to that of resected hearts. We conclude that apical resection in neonatal mice induces cardiomyocyte cell cycle activity and neomyogenesis, although scarring can occur. Surgical technique and definition of approach to assessing the extent of regeneration are both critical when using the neonatal mouse apical resection model.

Identification of Hypertension Susceptibility Loci on Rat Chromosome 12

Previous studies have identified multiple blood pressure and renal disease quantitative trait loci located on rat chromosome 12. In the present study, we narrowed blood pressure loci using a series of overlapping Dahl salt-sensitive/Mcwi (SS)-12 Brown Norway (BN) congenic lines. We found that transferring 6.1 Mb of SS chromosome 12 (13.4–19.5 Mb) onto the consomic SS-12BN background significantly elevated blood pressure on 1% NaCl (146 ± 6 versus 127 ± 1 mm Hg; P<0.001) and 8% NaCl diets (178 ± 7 versus 144 ± 2 mm Hg; P<0.001). Compared with the SS-12BN consomic, these animals also had significantly elevated albumin (218 ± 31 versus 104 ± 8 mg/d; P<0.001) and protein excretion (347 ± 41 versus 195 ± 12 mg/d; P<0.001) on a 1% NaCl diet. Elevated blood pressure, albuminuria, and proteinuria coincided with greater renal and cardiac damage, demonstrating that SS allele(s) within the 6.1 Mb congenic interval are associated with strong cardiovascular disease phenotypes. Sequence analysis of the 6.1 Mb congenic region revealed 12 673 single nucleotide polymorphisms between SS and BN rats. Of these polymorphisms, 293 lie within coding regions, and 18 resulted in nonsynonymous changes in conserved genes, of which 5 were predicted to be potentially damaging to protein function. Syntenic regions in human chromosome 7 have also been identified in multiple linkage and association studies of cardiovascular disease, suggesting that genetic variants underlying cardiovascular phenotypes in this congenic strain can likely be translated to a better understanding of human hypertension.

Role of genetic modifiers in an orthologous rat model of ARPKD.

Human data and animal models of autosomal recessive polycystic kidney disease (ARPKD) suggest that genetic factors modulate the onset and severity of the disease. We report here for the first time that ARPKD susceptibility is attenuated by introgressing the mutated Pkhd1 disease allele from the polycystic kidney (PCK) rat onto the FHH (Fawn-Hooded Hypertensive) genetic background. Compared with PCK, the FHH.Pkhd1 strain had significantly decreased renal cyst formation that coincided with a threefold reduction in mean kidney weights. Further analysis revealed that the FHH. Pkhd1 is protected from increased blood pressure as well as elevated plasma creatinine and blood urea nitrogen levels. On the other hand, liver weight and biliary cystogenesis revealed no differences between PCK and FHH.Pkdh1, indicating that genes within the FHH genetic background prevent the development of renal, but not hepatic, manifestations of ARPKD. Microarray expression analysis of kidneys from 30-day-old PCK rats revealed increased expression of genes previously identified in PKD renal expression profiles, such as inflammatory response, extracellular matrix synthesis, and cell proliferation genes among others, whereas the FHH.Pkhd1 did not show activation of these common markers of disease. This newly developed strain can serve as a tool to map modifier genes for renal disease in ARPKD and provides further insight into disease variability and pathophysiology.

Refined Mapping of the Renal Failure Rf-3 Quantitative Trait Locus

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.

SORCS1 contributes to the development of renal disease in rats and humans.

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.

A 4.1-Mb Congenic Region of Rf-4 Contributes to Glomerular Permeability

The combined transfer of two renal function quantitative trait loci (QTLs), Rf-1 (rat chromosome 1) and Rf-4 (rat chromosome 14), from the Fawn-hooded hypertensive rat onto the August Copenhagen Irish genetic background significantly increases proteinuria and demonstrates an interaction between these QTLs. Because the original Rf-4 congenic region is 61.9 Mbp, it is necessary to reduce this interval to feasibly search for variants responsible for renal susceptibility in this region. Here, we generated a minimal congenic line (Rf-1a+4_a) to identify a 4.1-Mb region of the Rf-4 QTL that significantly contributes to the severity of proteinuria in the Fawn-hooded hypertensive rat. Rf-1a+4_a animals have an increased glomerular permeability to albumin without significant changes in BP, indicating that at least one genetic element in this refined region directly affects renal function. Sequence analysis revealed no variants predicted to damage protein function, implying that regulatory elements are responsible for the Rf-4 phenotype. Multiple human studies, including recent genome-wide association studies, link the homologous human region with susceptibility to renal disease, suggesting that this congenic line is an important model for studying pathways that contribute to the progression of kidney disease.

IL13 Promotes in vivo Neonatal Cardiomyocyte Cell Cycle Activity and Heart Regeneration

There is great interest in identifying signaling mechanisms by which cardiomyocytes (CMs) can enter the cell cycle and promote endogenous cardiac repair. We have previously demonstrated that IL-13 stimulated cell cycle activity of neonatal CMs in vitro. However, the signaling events that occur downstream of IL-13 in CMs and the role of IL-13 in CM proliferation and regeneration in vivo have not been explored. Here, we tested the role of IL-13 in promoting neonatal CM cell cycle activity and heart regeneration in vivo and investigated the signaling pathway(s) downstream of IL-13 specifically in CMs. Compared with control, CMs from neonatal IL-13 knockout (IL-13-/-) mice showed decreased proliferative markers and coincident upregulation of the hypertrophic marker brain natriuretic peptide ( Nppb) and increased CM nuclear size. After apical resection in anesthetized newborn mice, heart regeneration was significantly impaired in IL-13-/- mice compared with wild-type mice. Administration of recombinant IL-13 reversed these phenotypes by increasing CM proliferation markers and decreasing Nppb expression. RNA sequencing on primary neonatal CMs treated with IL-13 revealed activation of gene networks regulated by ERK1/2 and Akt. Western blot confirmed strong phosphorylation of ERK1/2 and Akt in both neonatal and adult cultured CMs in response to IL-13. Our data demonstrated a role for endogenous IL-13 in neonatal CM cell cycle and heart regeneration. ERK1/2 and Akt signaling are important pathways known to promote CM proliferation and protect against apoptosis, respectively; thus, targeting IL-13 transmembrane receptor signaling or administering recombinant IL-13 may be therapeutic approaches for activating proregenerative and survival pathways in the heart. NEW & NOTEWORTHY Here, we demonstrate, for the first time, that IL-13 is involved in neonatal cardiomyocyte cell cycle activity and heart regeneration in vivo. Prior work has shown that IL-13 promotes cardiomyocyte cell cycle activity in vitro; however, the signaling pathways were unknown. We used RNA sequencing to identify the signaling pathways activated downstream of IL-13 in cardiomyocytes and found that ERK1/2 and Akt signaling was activated in response to IL-13.