Mutation of Plekha7 attenuates salt-sensitive hypertension in the rat Bradley T. Endresa,b, Jessica R. C. Priestleya, Oleg Palygina, Michael J. Flistera,b, Matthew J. Hoffmana, Brian D. Weinberga, Michael Grzybowskia,b, Julian H. Lombarda, Alexander Staruschenkoa, Carol Morenoa,b, Howard J. Jacoba,b,c, and Aron M. Geurtsa,b,d,1 Departments of aPhysiology and cPediatrics, bHuman and Molecular Genetics Center, and dCardiovascular Center, Medical College of Wisconsin, Milwaukee, WI 53226 Edited by Richard P. Lifton, Yale University School of Medicine, New Haven, CT, and approved July 23, 2014 (received for review June 24, 2014) PLEKHA7 (pleckstrin homology domain containing family A mem- hypertension and renal injury by selective ablation of adaptive im- em1Mcwi em1Mcwi ber 7) has been found in multiple studies as a candidate gene for mune cells in the SS-Rag1 and SS-Cd247 knockout human hypertension, yet functional data supporting this associa- rats (16, 17) and reduced hypertension and renal injury in the em1Mcwi tion are lacking. We investigated the contribution of this gene SS-Ncf2 (p67phox) null model exhibiting reduced medul- to the pathogenesis of salt-sensitive hypertension by mutating lary oxidative stress (18). Additionally, we have recently demon- Plekha7 in the Dahl salt-sensitive (SS/JrHsdMcwi) rat using zinc- strated multiple genes at a single hypertension GWAS-nominated finger nuclease technology. After four weeks on an 8% NaCl diet, locus (Agtrap-Plod1 locus) can have additive or subtractive effects homozygous mutant rats had lower mean arterial (149 ± 9 mmHg on blood pressure and renal function when mutated in the SS rat vs. 178 ± 7 mmHg; P < 0.05) and systolic (180 ± 7 mmHg vs. 213 ± (19). These previous studies highlight the utility of this model 8 mmHg; P < 0.05) blood pressure compared with WT littermates. system for testing the roles of GWAS candidate human disease Albumin and protein excretion rates were also significantly lower genes by disrupting their specific rat orthologs using ZFN tech- nology (20). in mutant rats, demonstrating a renoprotective effect of the mu- A single-nucleotide polymorphism (SNP) (rs381815, minor tation. Total peripheral resistance and perivascular fibrosis in the Plekha7 allele frequency 0.26) in intron 1 of the pleckstrin homology GENETICS heart and kidney were significantly reduced in mutant domain containing family A member 7 (PLEKHA7) gene, was animals, suggesting a potential role of the vasculature in the at- identified by five independent GWAS to be associated with el- tenuation of hypertension. Indeed, both flow-mediated dilation evated systolic blood pressure and hypertension in multiple and endothelium-dependent vasodilation in response to acetyl- populations (5, 6, 8, 21, 22). The associated locus contains only cholinewereimprovedinisolatedmesenteric resistance arteries the PLEKHA7 gene (5); however, the genetic mechanism(s) of Plekha7 mutant rats compared with WT. These vascular underlying this locus have not yet been functionally character- improvements were correlated with changes in intracellular cal- ized. PLEKHA7 is highly expressed in the kidney and heart, cium handling, resulting in increased nitric oxide bioavailability where it may be involved in formation and maintenance of the in mutant vessels. Collectively, these data provide the first func- apical junction complex of epithelial cells (23). However, limited tional evidence that Plekha7 may contribute to blood pressure data on PLEKHA7 function are available to extrapolate its regulation and cardiovascular function through its effects on the potential role(s) in the pathogenesis of hypertension. Here we vasculature. used ZFN mutagenesis to obtain the first evidence to our knowl- edge in any model system that Plekha7 has a functional role in genetic | GWAS | adherens junction | physiology | endothelial cells several hypertension-associated phenotypes in the rat. We found that mutation of Plekha7 in the SS rat attenuated salt-induced ypertension is a complex disease that is characterized by Hincreased blood pressure, renal damage, and vascular dys- Significance function which collectively increase risk of atherosclerosis, stroke, heart disease, and renal failure in one-quarter of all Zinc-finger nuclease (ZFN)-mediated mutagenesis has now en- adults worldwide (1–3). Because there is strong evidence of abled researchers to manipulate specific genes to test their heritability in hypertension (2, 4, 5), considerable effort has been function in animal models other than mice. Applying ZFNs to put toward identifying novel candidate genes and their molecular rats, we can now test the role of specific human genome-wide mechanisms. Genome-wide association studies (GWAS) have association studies (GWAS)-nominated genes for hypertension identified many potential hypertension loci, which shed light on in a well-characterized hypertensive rat model, the Dahl salt- the genetic complexity of this disease (5–8) but have provided sensitive rat. This study provides the first functional evidence little mechanistic insight. As such, validation and elucidation of that the GWAS-nominated gene Plekha7 plays an essential role the functional roles and disease mechanisms for these gene can- in blood pressure regulation and cardiovascular function by didates are the next important challenges (4). modulating vascular function. Our results indicate that Plekha7 Because hypertension is a complex disease (i.e., multiple plays a role in the regulation of intracellular calcium, nitric variants of small effect sizes contributing to disease risk), we oxide bioavailability, and the response of the vasculature to hypothesized candidate gene targeting on a genetically sensitized increased flow. background would reveal functional role(s) of genetic disease modifiers. The Dahl salt-sensitive (SS) rat is an inbred genetic Author contributions: B.T.E., J.R.C.P., O.P., M.J.F., M.J.H., B.D.W., M.G., J.H.L., A.S., C.M., H.J.J., model of salt-sensitive hypertension that displays hypertension- and A.M.G. designed research; B.T.E., J.R.C.P., O.P., M.J.F., M.J.H., B.D.W., M.G., and C.M. induced renal damage, cardiac hypertrophy and vascular dys- performed research; B.T.E., J.R.C.P., O.P., M.J.F., B.D.W., J.H.L., A.S., C.M., H.J.J., and A.M.G. function (9–11). These phenotypes are induced by exposing SS contributed new reagents/analytic tools; B.T.E., J.R.C.P., O.P., M.J.F., J.H.L., C.M., and A.M.G. rats to a high-salt diet, which results in rapid induction of hy- analyzed data; and B.T.E., J.R.C.P., O.P., M.J.F., J.H.L., A.S., and A.M.G. wrote the paper. pertensive phenotypes that closely resemble salt-induced hyper- The authors declare no conflict of interest. tension seen in humans (12–15). Knockout of specific genes in This article is a PNAS Direct Submission. this disease model using zinc-finger nuclease (ZFN) technology 1To whom correspondence should be addressed. Email: [email protected]. have revealed the importance of key mechanisms contributing This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. to hypertension risk, such as the protection from salt-induced 1073/pnas.1410745111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1410745111 PNAS | September 2, 2014 | vol. 111 | no. 35 | 12817–12822 Downloaded by guest on September 27, 2021 hypertension, reduced renal damage, and improved cardiac SS-Plekha7 mutant rats on a 0.4% NaCl diet but was significantly function. We also show that Plekha7 modulates calcium handling lower in the mutant rats on 8% NaCl diet compared with WT and nitric oxide (NO) bioavailability, both of which are required (Fig. S2A). We found no differences in BP variability between for normal vascular health. Collectively, these studies provide WT and mutant rats. Additionally, heart rate (HR) and HR significant mechanistic insight to the role of Plekha7 in salt- variability were not different between WT and mutant rats fed sensitive hypertension. a 0.4% NaCl diet or an 8% NaCl diet (Fig. S2B). Cumulatively, these data suggest that mutation of Plekha7 attenuates the de- Results velopment of salt-sensitive hypertension in the SS rat model. Targeting Plekha7 for Mutation Using ZFNs. To investigate the role of Plekha7 in hypertension, we used ZFNs to target genomic SS-Plekha7 Mutant Rats Have Reduced Renal Damage Compared with sequences encoding the pleckstrin homology (PH) functional WT Littermates. To examine whether renal damage under path- domain of Plekha7 in the SS rat (Fig. S1A). Injection of ZFNs ogenic conditions was affected by Plekha7 mutation, urine was targeting exon 8 of the Plekha7 gene into SS embryos resulted in collected and analyzed in male 9-wk-old WT and mutant rats a 19-bp deletion mutation in the coding sequence. This frame-shift (n = 10 per group) that were initially fed a 0.4% NaCl diet and mutation is predicted to cause the translation of 18 nonsense then switched to an 8% NaCl diet for a total of 28 d. Urinary amino acids and introduce a premature stop codon, yielding a albumin and protein excretion did not differ on the 0.4% NaCl truncated, nonfunctional protein consisting of 231 amino acids that diet (Fig. 2). After 28 d of the 8% NaCl diet, mutant rats had lacks the full PH domain (Fig. S1D). Verification of the mutation significantly lower urinary excretion of albumin (110 ± 10 mg/d; in homozygous SS-Plekha7 mutant animals was confirmed in P < 0.05) and protein (190 ± 16 mg/d; P < 0.05) compared with cDNA by RT-PCR and sequencing (Fig. S1 B and C). Quantitative WT levels of albuminuria (211 ± 22 mg/d) and proteinuria PCR revealed an approximated 50% reduction in Plekha7 ex- (381 ± 31 mg/d) (Fig. 2). In contrast, no differences in urine flow pression within the mutant animals compared with WT, indicating rate, urinary sodium, or urinary potassium concentrations be- nonsense-mediated decay of the transcript in the mutant rats (Fig.