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View of the 572 Location Demonstrates That the Amino Acid Mutation P.Lys572gln (K572Q) Destabilizes the Region from Amino Acids 547–596

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View of the 572 Location Demonstrates That the Amino Acid Mutation P.Lys572gln (K572Q) Destabilizes the Region from Amino Acids 547–596 BASIC RESEARCH www.jasn.org A Mutation in g-Adducin Impairs Autoregulation of Renal Blood Flow and Promotes the Development of Kidney Disease Fan Fan,1 Aron M. Geurts,2 Mallikarjuna R. Pabbidi,1 Ying Ge,1 Chao Zhang,1 Shaoxun Wang,1 Yedan Liu,1 Wenjun Gao,1 Ya Guo,1 Longyang Li ,1 Xiaochen He,1 Wenshan Lv,1 Yoshikazu Muroya,1 Takashi Hirata,1 Jeremy Prokop,3 George W. Booz,1 Howard J. Jacob,2 and Richard J. Roman1 1Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, Mississippi; 2Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin; 3Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan ABSTRACT Background The genes and mechanisms involved in the association between diabetes or hypertension and CKD risk are unclear. Previous studies have implicated a role for g-adducin (ADD3), a cytoskeletal protein encoded by Add3. BASIC RESEARCH Methods We investigated renal vascular function in vitro and in vivo and the susceptibility to CKD in rats with wild-type or mutated Add3 and in genetically modified rats with overexpression or knockout of ADD3. We also studied glomeruli and primary renal vascular smooth muscle cells isolated from these rats. Results This study identified a K572Q mutation in ADD3 in fawn-hooded hypertensive (FHH) rats—a mutation previously reported in Milan normotensive (MNS) rats that also develop kidney disease. Using molecular dynamic simulations, we found that this mutation destabilizes a critical ADD3-ACTIN binding site. A reduction of ADD3 expression in membrane fractions prepared from the kidney and renal vascular smooth muscle cells of FHH rats was associated with the disruption of the F-actin cytoskeleton. Compared with renal vascular smooth muscle cells from Add3 transgenic rats, those from FHH rats had elevated membrane expression of BKa and BK channel current. FHH and Add3 knockout rats exhibited impairments in the myogenic response of afferent arterioles and in renal blood flow autoregulation, which were rescued in Add3 transgenic rats. We confirmed these findings in a genetic complementation study that involved crossing FHH and MNS rats that share the ADD3 mutation. Add3 transgenic rats showed attenuation of proteinuria, glomerular injury, and kidney fibrosis with aging and mineralocorticoid-induced hypertension. Conclusions This is the first report that a mutation in ADD3 that alters ACTIN binding causes renal vascular dysfunction and promotes the susceptibility to kidney disease. JASN 31: 687–700, 2020. doi: https://doi.org/10.1681/ASN.2019080784 Hypertension and diabetes are the leading risk factors for CKD, but the genes and mechanisms Received August 6, 2019. Accepted December 14, 2019. involved are not well understood. Genome-wide Published online ahead of print. Publication date available at association studies (GWAS) have revealed multiple www.jasn.org. quantitative trait loci (QTL) that enhance the risk 1–6 Correspondence: Dr. Richard J. Roman, Department of Phar- of diabetic and hypertension nephropathy. macology and Toxicology, University of Mississippi Medical Knockout (KO) studies in mice and zebrafish Center, 2500 North State Street, Jackson, MS 39216. Email: have confirmed that some of the candidate genes [email protected] can alter renal function.7 However, none of the Copyright © 2020 by the American Society of Nephrology JASN 31: 687–700, 2020 ISSN : 1046-6673/3104-687 687 BASIC RESEARCH www.jasn.org human sequence variants have been shown to alter the expres- Significance Statement sion or function of the candidate proteins and cause renal disease in a transgenic model.7,8 The genes and mechanisms underlying the association between Genetic studies have also identified many regions of diabetes or hypertension and CKD risk are unclear. The authors fi Add3 thegenomethatinfluence the susceptibility to renal dis- identi ed a recessive K572Q mutation in g-adducin ( ), which encodes a cytoskeletal protein (ADD3), in fawn-hooded hyperten- 9 ease in rodent models of hypertension and diabetes. Many sive (FHH) rats—a mutation also reported in Milan normotensive candidate genes have been studied. However, only a poly- (MNS) rats that develop renal disease. They demonstrated that FHH morphism that prevents transcription of Rab38,which and Add3 knockout rats had impairments in the myogenic response blocksthereuptakeoffiltered albumin,10 has been con- of afferent arterioles and in renal blood flow autoregulation, which Add3 fi firmed to produce proteinuria in fawn-hooded hypertensive were rescued in transgenic rats. They con rmed the K572Q mutation’s role in altering the myogenic response in a genetic (FHH) rats. complementation study that involved crossing FHH and MNS rats. Previously, studies identified a QTL on chromosome 1 The work is the first to demonstrate that a mutation in ADD3 that (Rf-1), which is associated with proteinuria and glomerulo- causes renal vascular dysfunction also promotes susceptibility to sclerosis in FHH rats.9,11–13 In subsequent studies, we demon- kidney disease. strated that the FHH rat exhibits impaired myogenic response 14,15 and autoregulation of renal and cerebral blood. Transfer https://rgd.mcw.edu/rgdweb/report/gene/main.html?id5 of a portion of the Rf-1 region, including g-adducin (Add3), 2043. The K572Q ADD3 mutation in our colonies was also restored renal hemodynamics and attenuated proteinuria in validated by Sanger sequencing of PCR products using forward BN 14 fi an FHH.1 congenic strain. We also identi ed sequence primer 59-CATGTGCTGCAGGTCCGTTTATG-39 and reverse 14 fi variants in Add3 in FHH rats and con rmed that knockdown primer 59-CTGAGCAGAGCAGGTCCCTCTG-39. of ADD3 expression impaired the myogenic response of renal and cerebral arteries.16 Generation of FHH.Add3K572 Transgenic Rats Genetic KO studies only establish that loss of a candidate A full-length rat WT Add3 cDNA obtained from an expression gene has the potential to affect a phenotype. They provide no plasmid pCMV6-entry-Add3 purchased from Origene (Rock- information as to whether a sequence variant alters function. ville, MD) was inserted in a sleeping beauty transposon vector. Validation of a causal variant requires demonstration that ex- The expression of WT ADD3 in the transposon vector driven pression of the wild-type (WT) protein restores function in a by a CAG promoter was first validated in a cell culture system, BN transgenic or congenic strain. Therefore, we created FHH.1 and then, the construct was injected into the pronucleus of congenic and Add3 knock-in transgenic rats on the FHH ge- oocytes collected from female FHH rats along with SB100 BN netic background and Add3 KO rats on the FHH.1 and transposase mRNA as we previously reported.17 Transposon Sprague Dawley genetic backgrounds to evaluate the role of insertion sites were detected by ligation-mediated PCR.17 A the ADD3 mutation in altering renal hemodynamics and pro- single-transgene insertion was identified on chromosome moting CKD. We also performed a genetic complementation 10, which is located .64 kbp away from the protein shisa-6 study in an F1 cross of FHH and Milan normotensive (MNS) homolog precursor at its 59 end and .360 kbp away from rats that share the ADD3 mutation. the phosphoinositide-interacting protein at the 39 end. Con- firmation of the insertion site was verified by genotyping each animal using a Tri-Primer PCR strategy as we reported METHODS previously.17 Heterozygous founders were intercrossed to derive a homozygous transgenic line that was used for all Animal Models experiments. Experiments were conducted on male rats from colonies maintained at the University of Mississippi Medical Center Generation of KO Rats (UMMC). The original FHH (FHH/EurMcwi), Milan hyper- Zinc-finger nuclease (ZFN) technology18,19 was used to KO tension (MHS), and MNS breeders were obtained from the Add3 in both the FHH.1BN congenic and Sprague Dawley Medical College of Wisconsin (MCW). The FHH.1BN congenic strain backgrounds. A ZFN targeting the sequence ACCCGA [FHH.1BN-(D1Rat09-D1Rat225)/Mcwi] rats were generated at the UMMC. CTGAGGTGCtggagaAGAGAAATAAGATTCGGGA in exons The FHH.Add3K572 transgenic [FHHTg(CAG-Add3K572)Mcwi], 11 and 12 of the rat Add3 gene was designed and obtained FHH.1BN.Add3 KO, and Sprague Dawley.Add3 KO strains from Sigma-Aldrich (St. Louis, MO). The ZFN mRNA was were generated at the MCW and characterized at the injected into the pronucleus of fertilized FHH.1BN and Spra- UMMC. The FHH 3 FHH.1BN and FHH 3 MNS F1 crosses gue Dawley embryos and transferred to the oviduct of pseu- were bred at the UMMC. All protocols were approved by the dopregnant females. Founders were identified using a Cel-1 Institutional Animal Care and Use Committees at the UMMC assay.20 PCR genotyping of tail biopsies from the founders and the MCW. Sequence variants in Add3 in different strains confirmed 68- and 14-bp deletions in FHH.1BN and Sprague were aligned to Brown Norway (BN) reference genome and Dawley genetic background, respectively, using forward identified using the Genome Analysis Tool kit available at primer 59-GCCCCCATGAGTCACTACAC-39 and reverse 688 JASN JASN 31: 687–700, 2020 www.jasn.org BASIC RESEARCH primer 59-GCTACAGGAAGCATCTCCTGTG-39.Founders Biotechnology) as previously described,16 and b-actin was with Add3 deletion were backcrossed to the parental strain used as a loading control. to generate heterozygous F1 rats. Heterozygous F1 siblings were then intercrossed to derive a homozygous KO line used Patch-Clamp Studies for all experiments. VSMCs isolated from renal microvessels obtained from FHH, FHH.Add3, FHH.1BN congenic, and FHH.1BN Add3 Immunocytochemistry KO rats were used for patch-clamp studies as we previously Vascular smooth muscle cells (VSMCs) were isolated from described.16 Whole-cell currents were recorded before and pooled renal microvessels isolated from FHH and FHH.Add3 after blockade of the BK channel with iberiotoxin (IBTX; 2 rats as we described previously.16,21 Briefly, renal microvessels 10 7 M) using an Axopatch 200B amplifier (Axon Instru- were isolated using a sieving procedure and washed with ice- ments, Foster City, CA).
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