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). Clampfitsoftware(version10.0; coldphysiologicsaltsolution(PSS)containing(pH7.4) Axon Instruments) was used for data acquisition and analy- 119mMNaCl,4.7mMKCl,1.6mMCaCl2,1.2mM sis. Differences in the size of the VSMCs were normalized MgSO4, 18 mM NaHCO3,1.2mMNaH2PO4, 10 mM glucose, by the expression of peak current (in picoamperes) as a cur- 0.03 mM EDTA, and 5 mM HEPES. The vessels were incu- rent density (picoamperes per picrofarads). bated in PSS supplemented with papain (22.5 U/ml) and di- thiothreitol (2 mg/ml) at 37°C for 15 minutes with gentle Examination of BP and Proteinuria rotation; then, they were pelleted and resuspended in PSS Mean arterial pressure (MAP) detected by telemetry and pro- supplemented with collagenase (250 U/ml), trypsin inhibitor teinuria collected in metabolic cages was measured at 3-week (10,000 U/ml), and elastase (2.4 U/ml) and incubated at 37°C intervals in FHH and FHH.Add3K572 transgenic rats from with gentle rotation for another 15 minutes. After centrifuga- 12 to 22 weeks or weekly before and after induction of deox- tion, single cells were released into DMEM (Thermo Fisher ycorticosterone (DOCA) salt hypertension for 3 weeks. Scientific, Waltham, MA) supplemented with 20% FBS and Hypertension was induced in uninephrectomized rats by im- antibiotics and seeded in chamber slides that were precoated plantation of a 21-day release DOCA pellet (200 mg) with 1% with Cell-Tak (Agilent, Santa Clara, CA). Immunocytochem- sodium chloride drinking water. Urine protein concentrations istry was performed immediately on freshly isolated cells using were determined using the Bio-Rad Protein Assay buffer (Bio- primary antibodies against KCNMA1 (APC-107, 1:50; Alo- Rad Laboratories, Hercules, CA). mone Labs, Jerusalem, Israel) and Adducin g (sc-25733; Santa Cruz Biotechnology, Santa Cruz, CA) followed by incubation The Myogenic Response of the Afferent Arteriole with Alexa Fluor–labeled secondary antibodies (Thermo Glomerulus-attached afferent arterioles (Af-arts) were micro- Fisher Scientific). The slides were covered with an antifade dissected and transferred to a temperature-controlled cham- mounting medium with DAPI (Vector Laboratories, Burlin- ber. The Af-arts were cannulated using concentric pipettes, game, CA). Images were obtained using a Nikon Eclipse 55i and the myogenic responses were determined by measuring microscope connected with a DS-FiL1 color camera (Nikon, the inner diameters of the Af-art in response to an elevation in Melville, NY). Isolated VSMCs were also cultured with 20% perfusion pressures from 60 to 120 mm Hg using a digital FBS DMEM on Cell-Tek–coated dishes. Early-passage (P2) CCD camera (Andor, Concord, MA) on an inverted micro- VSMCs were incubated with the Adducin g first antibody fol- scope. The data were analyzed using NIS-Elements software lowed by Alexa Fluor–labeled secondary antibody or Alexa (Nikon) as we previously reported.16,22 Fluor–labeled Phalloidin for F-actin (Thermo Fisher Scien- tific) staining. Cells were imaged using a Nikon C2 laser scan- Autoregulation of Renal Blood Flow ning confocal inverted microscope. Renal blood flow (RBF) autoregulation was compared in 9- to 12-week-old rats as previously described.23 Briefly, the rats Western Blot were anesthetized with ketamine (30 mg/kg intramuscularly) The renal cortex and freshly isolated primary VSMCs were and Inactin (50 mg/kg intraperitoneally). RBF was measured homogenized in radioimmunoprecipitation assay buffer with a transonic flow probe (Transonic Systems Inc., Ithaca, (Sigma-Aldrich) supplemented with protease and phos- NY) placed around the left renal artery. BP was measured via phatase inhibitors (Thermo Fisher Scientific). Membrane catheters implanted in the carotid and femoral arteries, and fractions were obtained by centrifugation at 11,000 3 g for renal perfusion pressure (RPP) was adjusted with clamps 15 minutes at 4°C. Aliquots of the renal cortical homogenates placed on the abdominal aorta above and below the left renal or kidney and VSMC membrane fractions (75 mg for the renal artery. The left kidney was harvested for western blot or im- cortex and 35 mg for the kidney membrane and VSMC mem- munohistochemistry at the end of the experiment. branes) were electrophoresed on SDS-polyacrylamide gels, transferred to nitrocellulose membranes with a Trans- Glomerular Capillary Pressure Blot Turbo Transfer System (Bio-Rad, Hercules, CA), and in- Glomerular capillary pressure (Pgc) was measured in 9- to cubated with an anti-Add3 antibody (sc-365177; Santa Cruz 12-week-old FHH and FHH.1BN congenic rats as previously

JASN 31: 687–700, 2020 ADD3, vascular dysfunction and CKD 689 BASIC RESEARCH www.jasn.org described.24 Briefly, rats were anesthetized with ketamine RESULTS (30 mg/kg intramuscularly) and Inactin (50 mg/kg intraperitoneally). Catheters were placed in the femoral artery Identification of a p.Lys572Gln (K572Q) Mutation in and jugular vein for the measurement of arterial pressure ADD3 in the FHH and MNS Rats and intravenous infusion. Pgc was estimated by measuring We identified a p.Lys572Gln (K572Q) mutation in ADD3 in FHH stop-flow pressures in proximal tubules using a servo null rats. The same mutation was previously reported by Tripodi et al.28 micropressure device (model 900; World Precision Instru- in MNS rats, which are also susceptible to kidney injury. The K572 ments, Sarasota, FL). allele in ADD3 was reported to be expressed in 13 normotensive strains, including Sprague Dawley, BN, and August Copenhagen Glomerular Permeability to Albumin Irish, and five hypertensive strains, including MHS and spontane- These experiments were performed in 9- to 12-week-old Spra- ously hypertensive rats (SHRs).28 Amino acid 572 is located in gue Dawley, FHH, and FHH.Add3K572 transgenic rats. High exon 13 at a critical ADD3-ACTIN interaction site (Figure 1A). molecular mass (500 kD) FITC-Dextran dissolved in saline The ADD3 mutation destabilizes a region from amino acids was injected into anesthetized rats. Glomeruli were isolated, 547–596 (Figure 1, B and C). These changes are predicted to alter and permeability to albumin (Palb) was compared using a fluo- the ability of ADD3 to regulate the actin cytoskeleton, which rescent dilution technique as previously reported.25 The reflec- influences signal transduction and membrane trafficking.29 fi tion coefficient (sAlb) indicating the ratio of the change in We con rmed that ADD3 expression was reduced in membrane fluorescence intensity after rapidly reducing albumin concen- fractions prepared from the kidney (Figure 1D) and renal VSMCs tration in the bath (6%–4%) to the expected change (33%) in (Figure 1E) of FHH rats. ADD3 redistributed from the cell mem- glomerular volume in response to the decrease in oncotic pressure brane to the cytoplasm of freshly isolated (Figure 1F) and primary was examined. The convective permeability to albumin (1 2sAlb) cultured renal VSMCs (Figure 1G) in FHH versus FHH.Add3 rats. was compared as an index of the relative degree of glomerular injury between strains in populations of glomeruli.25,26 Validation of ADD3 Protein Expression in the Newly Generated Rat Models Assessment of Renal Injury The Add3 transgenic and KO rat models were created on the FHH, The kidneys were fixed in 10% buffered formalin solution, and FHH.1BN, and Sprague Dawley genetic backgrounds. The FHH rat paraffin sections (3 mm) were prepared and stained with Mas- expresses the Q572 allele. The FHH.1BN congenic rat has a 2.4- son trichrome or hematoxylin-eosin. Thirty glomeruli per rat Mbp region of chromosome 1, including Add3,fromBNratsin- were evaluated for the degree of glomerular injury as previ- trogressed onto the FHH background. Both FHH.1BN congenic ously described.27 The degree of glomerular and renal inter- and Sprague Dawley rats carry the reference K572 allele of ADD3. stitial fibrosis was examined and calculated by the measuring Transgenic FHH-Tg(CAG-Add3K572) Mcwi rats (referred to as percentage of blue collagen staining using the NIS-Elements D FHH.Add3)werecreatedusingaSleeping Beauty transposon trans- 4.6 software (Nikon). Areas that exhibited red fluorescence at genic technique17 to overexpress the reference ADD3 allele (K572) the corticomedullary junction were also analyzed to access the in FHH rats (Supplemental Figure 1A). ZFN technology18,19 was formation of protein casts. used to KO the reference K572 allele of ADD3 in both the Paraffin sections were deparaffinized with xylene and rehy- FHH.1BN congenic and Sprague Dawley strains that do not express drated with ethanol as we previously described.27 The sections ADD3 (Supplemental Figure 1, B and C). We refer to these strains were then permeabilized followed by antigen retrieval and as FHH.1BN Add3 KO and Sprague Dawley.Add3 KO, respectively. incubated with antibody to nephrin (1:50; Fitzgerald, Wash- ington, DC) and Alexa Fluor–labeled secondary antibody. Effects of ADD3-K572Q Variant on the F-Actin Endogenous fluorescence was quenched with 0.005% Evans Cytoskeleton and IBTX-Sensitive BK Channel Current in blue. The slides were cover slipped using an antifade mounting VSMCs Isolated from FHH Rats medium with DAPI (Vector Laboratories), and the images IBTX-sensitive BK peak current densities were higher in VSMCs were captured using a Nikon Eclipse 55i microscope equipped of FHH and FHH.1BN Add3 KO rats than controls (Figure 2, A with a DS-Fil1 color camera (Nikon). Mean fluorescence in- and B). The expression of pore-forming BK a-subunits was tensities for nephrin staining per glomerulus were analyzed elevated in the plasma membrane of renal VSMCs of FHH ver- using the NIS-Elements D 4.6 software (Nikon) as we previ- sus FHH.Add3K572 rats (Figure 2C). The actin cytoskeleton was ously described.27 altered in primary cultured VSMCs isolated from FHH rats with the loss of F-actin stress filaments and the formation of a more Statistical Analyses branched F-actin network (Figure 2D). All data are presented as mean values 6SEM. A two-way repeated measures ANOVAwas used to compare the significance Effects of ADD3-K572Q Variant on the Myogenic of differences in corresponding values between groups fol- Response of the Af-art and Autoregulation of RBF lowed by a Holm–Sidak test. A P value ,0.05 was considered The diameter of the Af-art decreased by 12.1%60.8% in Spra- to be significant. gue Dawley rats (carrying K572 allele) when perfusion

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AB C ADD3 mds ADD3 mds

10 K572 Q572 15 K572 Q572

K572 8 10 6

4 ACTIN 5 2 ADD3 Carbon Alpha RMSF (A) Carbon Alpha RMSD (A) 0 0 1 0 2 4 6 8 10 12 14 16 18 20 7 81 87 161 321 167 401 561 247 241 481 641 Time (ns) 327 D ADD3 ACTIN FHH FHH.1BN

Add3 15 K572 Q572 β-Actin 10

E 5 FHH FHH.Add3 Add3 0 Carbon Alpha RMSF (A) β-Actin 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 Amino Acid F G

Figure 1. A p.Lys572Gln (K572Q) mutation in the ADD3 protein alters the actin binding site in the FHH rat. (A) Model of ADD3 (cyan) docked to ACTIN (gray) using HADDOCK web server followed by energy minimization indicates that the location of K572 (red) is at a critical ADD3-ACTIN binding site. (B) ADD3 protein models containing K572 or Q572 allele were run for 20 ns of molecular dynamics simulations (mds) using the AMBER03 force field to reach dynamic equilibrium. (C) The individual amino acid movement was assessed for both ADD3 and ACTIN, and a zoomed-in view of the 572 location demonstrates that the amino acid mutation p.Lys572Gln (K572Q) destabilizes the region from amino acids 547–596. (D) Representative western blot indicating the expression of an immunoreactive band at the apparent molecular mass of ADD3 (94 kD) in the membrane fraction of the kidney of FHH.1BN congenic but not in FHH rats; b-actin was used as a loading control. (E) ADD3 was detected in the membrane fraction of primary renal VSMCs isolated from FHH.Add3 transgenic rats that carry the reference K572 allele, but it was barely detectable in FHH rats; b-actin was used as a loading control. Distribution of ADD3 in the membrane of (F) freshly isolated and (G) cultured primary renal VSMCs of FHH rats is reduced relative to FHH.Add3 transgenic rats. White lines indicate the outline of the cells. pressure was increased, and RBF only increased by 3.5%60.9% FHH 3 MNS rats (Figure 4). RBF increased by 7.3%61.7% when RPP was elevated from 100 to 150 mm Hg (Figure 3A). in FHH 3 FHH.1BN versus 33.1%64.4% in FHH 3 MNS Both FHH.1BN congenic and MHS rats displayed a normal myo- rats when RPP was elevated from 100 to 150 mm Hg. The genic response and autoregulation. However, the Af-art failed recessive inhibitory effect of Q572 ADD3 was confirmed in to constrict in FHH and MNS rats, and RBF increased by cerebral arteries isolated heterozygous or homozygous Sprague 38.0%63.0% when RPP was increased. The renal vascular im- Dawley.Add3 KO rats (Supplemental Figure 2). These results pairment was rescued in FHH.1BN congenic and FHH.Add3K572 demonstrate that one copy of the reference Add3 K572 allele is transgenic rats (Figure 3B). KO of Add3 abolished the myo- sufficient to maintain the myogenic response and provides genic response and RBF autoregulation in FHH.1BN congenic strong genetic evidence that the mutant ADD3 underlies renal (Figure 3C) and Sprague Dawley rats (Figure 3D). vascular dysfunction in FHH and MNS rats.

Genetic Complementation Studies Effects of ADD3-K572Q Variant on MAP and Renal Injury The Af-art constricted in an F1 cross of FHH 3 FHH.1BN The consequence of the ADD3 mutation in FHH rats on rats when perfusion pressure was increased but dilated in the development of CKD with aging and hypertension was

JASN 31: 687–700, 2020 ADD3, vascular dysfunction and CKD 691 BASIC RESEARCH www.jasn.org

A B 180 FHH (14) 180 FHH (14) FHH.Add3 (11) FHH.Add3 (11) FHH.1BN (13) FHH.1BN (13) 150 150 FHH.1BN.Add3 KO (10) FHH.1BN.Add3 KO (10) FHH + IBTX (14) 120 FHH.Add3 + IBTX (11) 120 FHH.1BN + IBTX (13) FHH.1BN.Add3 KO + 90 IBTX (10) 90 *

60 IBTX-Sensotive 60 * 30 † 30 * Peak Current Densities (pA/pF) * † Peak Current Densities (pA/pF) 0 0 -60 -40 -20 0 20 40 60 80 100 120 -60 -40 -20 0 20 40 60 80 100 120 -30 -30 Membrane Potential (mV) Membrane Potential (mV)

Figure 2. A p.Lys572Gln (K572Q) mutation in the ADD3 protein in the FHH rat is associated with elevated IBTX-sensitive potassium (BK) channel current and disruption of the F-actin cytoskeleton in VSMCs. (A and B) Comparison of IBTX-sensitive potassium channel current densities in VSMCs isolated from renal vasculature of FHH, congenic, Add3 transgenic, and KO rats. Numbers in parentheses † indicate the number of animals studied. *Significant differences compared with the corresponding values in the WT strains; significant differences before and after IBTX within the same. (C) Immunocytochemistry demonstrating elevated expression of BK a-subunits in the plasma membrane of renal VSMCs isolated from FHH compared with FHH.Add3 rats. (D) The actin cytoskeleton was disrupted in primary cultured VSMCs isolated from FHH compared with FHH.Add3 rats. evaluated. MAP was similar in FHH and FHH.Add3K572 trans- rats as previously reported.25 Overexpression of ADD3K572 genic rats as they aged from 12 to 22 weeks (Figure 5A) and rescued the elevated Palb in FHH.Add3K572 transgenic rats after induction of DOCA/salt hypertension (Figure 5C). Pro- (Figure 6, B and C). Hypertensive FHH.Add3K572 rats exhibi- teinuria increased from 37.662.2 to 294.0616.0 mg/d in ted greater glomerular nephrin expression (Figure 6D, 12- versus 22-week-old FHH rats but rose to a lesser extent Supplemental Figure 3A), reduced glomerular injury, fibrosis in age-matched FHH.Add3K572 rats (Figure 5B). Similarly, pro- (Figure 6E, Supplemental Figure 3, B and C), and less protein tein excretion was lower in FHH.Add3K572 than FHH rats after cast formation (Figure 6F, Supplemental Figure 3D) than hy- the induction of DOCA/salt hypertension (Figure 5D). pertensive FHH rats.

Effects of ADD3-K572Q Variant on Pgc, Palb, and Renal Injury after Induction of Hypertension DISCUSSION Pgc increased by 15 mm Hg in FHH rats when RPP was increased versus only 4 mm Hg in the age-matched FHH.1BN Patients who are diabetic or hypertensive are at higher risk for congenic strain (Figure 6A). Using a fluorescence dilution the development of CKD.30 There is a significant genetic com- technique, we confirmed that Palb was elevated in FHH ponent because less than half of these individuals ever develop

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A B 120 SD (12) 120 SD (12) 160 SD (13) FHH (15) 160 FHH (15) FHH (15) FHH.1BN (27) FHH.1BN (27) FHH.1BN (21) 140 140 * † 110 MNS (6) * † 110 FHH.Add3 (10) FHH.Add3 (9) MHS (6) † † † * 120 * * 120 * † * 100 100 100 100

SD (13) 80 80 FHH (15) 90 90 * FHH.1BN (21) 60 60 MNS (12) MHS (4) 80 40 80 40 Diameter of Af-art (% 60 mmHg) Diameter of Af-art (% 60 mmHg) Renal Blood Flow (% of 100 mmHg) 60 120 Renal Blood Flow (% of 100 mmHg) 70 90 110 130 150 60 120 70 90 110 130 150 Perfusion Pressure (mmHg) Perfusion Pressure (mmHg) Perfusion Pressure (mmHg) Perfusion Pressure (mmHg)

C D 120 SD (12) 160 120 SD (12) 160 FHH (15) * † SD.Add3 KO (7) FHH.1BN (27) 140 † 140 110 FHH.1BN. * 110 Add3 KO3 (10) * † 120 120 * * † * 100 100 100 100

80 SD (13) 80 90 FHH (15) 90 60 FHH.1BN (21) 60 FHH.1BN. SD (13) Add3 KO (7) SD.Add3 KO (10) 80 40 80 40 Renal Blood Flow (% of 100 mmHg) Renal Blood Flow (% of 100 mmHg) Diameter of Af-art (% 60 mmHg) Diameter of Af-art (% 60 mmHg) 60 120 70 90 110 130 150 60 120 70 90 110 130 150 Perfusion Pressure (mmHg) Perfusion Pressure (mmHg) Perfusion Pressure (mmHg) Perfusion Pressure (mmHg)

Figure 3. The ADD3-K572Q variant impairs the myogenic response of the Af-art and autoregulation of RBF. (A) The diameter of the renal Af-art in rats carrying K572 allele, including Sprague Dawley, FHH.1BN congenic, and MHS rats, decreased when perfusion pressure increased from 60 to 120 mm Hg (left panel). RBF was well autoregulated when RPP elevated from 100 to 150 mm Hg in these strains (right panel). In contrast, Af-art did not constrict following elevations in pressure in FHH and MNS rats carrying Q572 allele, and RBF was poorly autoregulated when RPP was elevated from 100 to 150 mm Hg. (B) Expression of WT ADD3 protein in FHH.1BN congenic and FHH.Add3 transgenic rats rescued the Af-art myogenic response (left panel) and RBF autoregulation (right panel). (C) KO of Add3 in the FHH.1BN genetic background abolished the Af-art myogenic response (left panel) and RBF autoregulation (right panel). (D) KO of Add3 in Sprague Dawley rats also abolished the myogenic response of the Af-art (left panel) and RBF autoregulation (right panel). Numbers in parentheses indicate the number of animals studied. *Significant difference compared with the corresponding † value in the WT strains; significant difference from the corresponding value in Sprague Dawley rats. renal disease.31,32 GWAS have identified .100 regions of the mutation in the Rab38 gene located in the Rf-2 region that genome that influence the susceptibility to CKD.33 However, contributes to proteinuria in FHH rats by attenuating tubular identification of the specific variants involved has proven to be reuptake of filtered protein.10,40 More recently, a variant in the difficult due to the heterogeneous patient populations, differ- Rab38 has been linked to proteinuria in patients who are di- ences in disease progression, and uncontrolled environmental abetic.4 However, introgression of the WT Rab38 gene in con- factors.31 genic or transgenic strains had no effect on the renal vascular Our group has been studying CKD susceptibility using in- function,10,40 suggesting other genes in the Rf-1 QTL must alter bred FHH rats, which reduces genetic heterogeneity and al- renal hemodynamics and contribute to CKD in FHH rats. lows for control of environmental influences.12,13,31,34 This WereportedthatsubstitutionofaportionoftheRf-1 re- strain is characterized by proteinuria, glomerulosclerosis, renal gion, including Add3, attenuated proteinuria and rescued interstitial fibrosis, and mild hypertension as they age.11,35–41 the impaired myogenic response and autoregulation of renal FHH rats are also more susceptible to hypertension-induced and cerebral blood flow14,15 in an FHH.1BN congenic strain. renal injury than control August Copenhagen Irish and We established Add3 as a candidate gene by showing that BN strains.39–41 We previously reported that FHH rats do not knockdown of its expression impaired the myogenic re- autoregulate RBF due to impaired myogenic response in renal sponse of renal and cerebral arterioles ex vivo.16 We con- arterioles.14,16,42 Five QTLs (Rf-1 to Rf-5) for proteinuria have firmed that FHH and MNS rats, which are both susceptible been identified in this strain.31 Two of these loci (Rf-1 and Rf-2) to renal injury,42– 44 share the mutant Q572 locus in are on rat chromosome 1.40 We subsequently identified a ADD3, whereas the reference K572 allele is expressed in

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A 120 SD (12) 160 FHH (15) FHH.1BN (27) 140 * † 110 FHH X 1BN (8) * † 120

100 100

80 90 SD (13) 60 FHH (15) Diameter of Af-art (% 60 mmHg) Renal Blood Flow (% of 100 mmHg) FHH.1BN (n=21) FHH X 1BN (7) 80 40 60 120 70 90 110 130 150 Perfusion Pressure (mmHg) Perfusion Pressure (mmHg)

B 120 160 SD (12) FHH (15) MNS (6) 140 † FHH X MNS (8) † 110 † † † 120 † 100 100

80 90 SD (13) 60 FHH (15) Diameter of Af-art (% 60 mmHg) Renal Blood Flow (% of 100 mmHg) MNS (12) FHH X MNS (9) 80 40 60 120 70 90 110 130 150 Perfusion Pressure (mmHg) Perfusion Pressure (mmHg)

Figure 4. A genetic complementation study confirms that a shared ADD3 mutation causes renal vascular dysfunction in FHH and MNS rats. (A) One copy of ADD3 WT K572 allele in an F1 cross of FHH and FHH.1BN congenic (FHH 3 1BN) rats restored the Af-art myogenic response (left panel) and RBF autoregulation (right panel) relative to FHH rats. (B) Two copies of ADD3 mutant Q572 allele in an F1 cross of FHH and MNS rat (FHH 3 MNS) abolished the Af-art myogenic response (left panel) and RBF autoregulation (right panel). Numbers in parentheses indicate the number of animals studied. *Significant difference compared with the corresponding value in the † WT strains; significant difference from the corresponding value in Sprague Dawley rats.

5 hypertensive strains and 13 of 18 normotensive strains se- ADD subunits are critical to its ability to cap F-actin and quenced in a previous study.28 prevent excess polymerization.29,47,48 Phosphorylation of Adducin is a cytoskeletal protein composed of heterodim- this region by protein kinases C and A dissociates ADD and ers of a- (ADD1) and b-subunits (ADD2) or a-and spectrin from the membrane and disrupts actin capping and g-subunits (ADD3).29 ADD3 is expressed in most tissues, in- the cytoskeleton.46 cluding blood vessels, podocytes, and distal tubules in the Our results indicate that the K572Q mutation in ADD3 kidney.16,29,45 These tetrameric proteins have similar struc- destabilizes the region of amino acids 547–596 at a critical tures, including a globular head, a neck region important ADD3-ACTIN interaction site. This region also contains three for dimerization, and a positively charged C terminus essential serine phosphorylation sites that regulate the function of this for dimerization and the association of adducin with actin, protein.49 The mutation is predicted to alter the conﬁguration spectrin, and the plasma membrane.29,46 The tails of the of ADD3, disassociate it from the membrane, and diminish its

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A C 200 FHH (8) 240 FHH (5) FHH.Add3 (10) 220 FHH.Add3 (12) 180 200 160 180

140 160 MAP (mmHg) MAP (mmHg) 140 120 120 100 100 12 14 16 18 20 22 0123 Age (weeks) DOCA Treatment (weeks)

B D 350 FHH (30) FHH (21) 600 300 FHH.Add3 (13) FHH.Add3 (10) 500 250 400 200 * 300 150 * 100 200

Proteinuria (mg/day) 50 100 Proteinuria (mg/day) 0 0 12 14 16 18 20 22 24 0123 Age (weeks) DOCA Treatment (weeks)

Figure 5. The ADD3-K572Q variant increases mean arterial pressure (MAP) and urine protein excretion with age and after induction of hypertension in FHH rats. (A) MAP was similar in 12- to 22-week-old FHH and FHH.Add3 transgenic rats. (B) Proteinuria was significantly lower in 12- to 22-week-old FHH.Add3 transgenic compared with FHH rats. (C) MAP rose to the same extent in FHH.Add3 transgenic versus FHH rats after induction of DOCA/salt hypertension. (D) Urine protein excretion was lower in hypertensive FHH.Add3 transgenic versus FHH rats. Numbers in parentheses indicate the number of animals studied. *Significant difference compared with the corresponding value in FHH rats. ability to regulate the actin cytoskeleton in VSMCs, which in a cross of FHH and FHH.1BN rats with one copy of the WT influences signal transduction and membrane trafficking.29 ADD3 allele. In addition, the impaired myogenic response in This prediction was confirmed by our finding of reduced Sprague Dawley.Add3 KO rats was restored when they were ADD3 expression in membrane fractions prepared from the crossed with Sprague Dawley rats carrying the WT K572 kidney and primary renal VSMCs of FHH rats. Consistent ADD3. These results indicate that the shared mutation in with our previous reports,16,50 we also found that ADD3 re- ADD3 plays a causal role in renal vascular dysfunction in distributed to a perinuclear location in freshly isolated and FHH and MNS rats. cultured primary VSMCs from FHH rats in association with An activating mutation in ADD1, but not Add3, has been the loss of F-actin stress filaments and the formation of a more linked to the development of hypertension,51–57 stroke,58 and branched F-actin network. cardiovascular dysfunction59–61 in human genetic association However, confirmation that the K572Q mutation is causal studies and the development of hypertension in MHS rats.52,62 requires demonstration that the expression of the WT protein Previous studies focused on the role of a G460W mutation restores function. In this regard, the myogenic response of in ADD1 in promoting hypertension by altering actin poly- the Af-art and RBF autoregulation was impaired in FHH merization and enhancing sodium transport in the kid- and MNS rats carrying the Q572 mutant allele and Add3 ney.52,54,63–66 However, there are now a few reports that KO rats on both Sprague Dawley and FHH.1BN genetic back- mutations in ADD3 are also linked to human disease. In this grounds. The response was intact in Sprague Dawley and regard, a rare variant in ADD3 found in three families was MHS rats, and it was rescued in FHH.1BN and FHH.Add3K572 associated with cognitive dysfunction and nephrotic syn- rats that express WT ADD3. drome in one of these families.67 A G367D mutation in The results of our genetic complementation study indi- ADD3 that impaired dimerization of ADD3 with ADD1, actin cated that the myogenic response of the Af-art and autoregu- capping, and disrupted the cytoskeleton in fibroblasts, was lation of RBF was impaired in an F1 cross of FHH and MNS associated with cerebral palsy in related families.68 Finally, rats that share the K572Q mutation in ADD3 but normalized SNPs near the Add3 gene have been linked to biliary atresia.69

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A D FHH (22) 65 FHH.1BN (32)

Pgc (mmHg) *

45 100 140 Perfusion Pressure (mmHg)

B SD (16, 288) E 110 FHH (12, 255) 100 FHH.Add3 (4, 119)

80 †

70 * % Fluorescence 60 0 30 60 90 120 150 Time (seconds)

C F 0.6 0.5 ) 0.4 Alb 0.3 * 0.2 Palb (1- δ 0.1 16, 288 12, 255 4, 119 0.0 SD FHH FHH.Add3

Figure 6. The ADD3-K572Q variant alters Pgc, Palb, and glomerular function in FHH rats after induction of hypertension. (A) Pgc estimated from measurements of stop-flow pressure in the early proximal tubules increased to a lesser extent from 49.061.0 to 53.161.6 mm Hg in 3-month FHH.1BN rats when RPP was increased from 100 to 140 mm Hg versus from 48.860.9 to 63.661.2 mm Hg in age-matched FHH rats. Numbers in parentheses indicate the number of glomeruli studied. (B and C) Fluorescence intensity changes (percentages) in glomeruli isolated from Sprague Dawley, FHH, and FHH.Add3 transgenic rats in response to a change in albumin concentration in the bath albumin concentration from 6% to 4% are presented. Palb is calculated as 1 2 sAlb in glomeruli isolated from Sprague Dawley, FHH, and FHH.Add3 transgenic rats; sAlb is the convective albumin reflection coefficient. Numbers in parentheses or bars indicate the number of animals and glomeruli studied. *Significant difference from the corresponding values in WT strains; † significant difference from the IBTX values in Sprague Dawley rats. (D) Glomerular nephrin expression is lower in FHH versus FHH.Add3 transgenic rats after 3 weeks of DOCA/salt hypertension. Quantitation is presented in Supplemental Figure 3A. (E) Glomerular injury and renal interstitial fibrosis were reduced FHH.Add3 transgenic versus FHH rats after 3 weeks of DOCA/salt hypertension. Quantitation is presented in Supplemental Figure 3, B and C. (F) Tubular protein cast formation (red stain) on hematoxylin-eosin–stained sections in hypertensive FHH.Add3 transgenic versus FHH rats. Quantitation is presented in Supplemental Figure 3D.

The mechanism by which loss of adducin function impairs activity could be secondary to disruption of the actin cytoskel- the myogenic response remains to be determined. We reported eton that increases its trafficking into the membrane or re- that the impaired myogenic response is associated with an duces recycling of the channel into endosomes. Alternatively, elevation in BK channel activity.14,15,70 Themyogenicre- changes in the actin cytoskeleton could affect the actin-myosin sponse could be restored by blocking the BK channel with contractile mechanism, the membrane expression of other ion IBTX. In this study, the expression of BK channels in the channels, or signaling mechanisms.29,48,50 membrane and channel activity was elevated in VSMCs iso- Hyperfiltration and increased Pgc are thought to contrib- lated from FHH rats. The elevated BK channel activity was ute to podocyte injury in salt-sensitive hypertensive and di- normalized by the expression of WT-ADD3 in FHH.1BN con- abetic models.71,72 In this study, we confirmed that Pgc and genic and FHH.Add3 transgenic strains. Elevated BK channel Palb were elevated in FHH rats. Renal injury was attenuated activity was also seen in ADD3 KO rats. Increased BK channel in FHH.Add3K572 rats with reduced protein excretion, less

696 JASN JASN 31: 687–700, 2020 www.jasn.org BASIC RESEARCH glomerular injury, and increased nephrin expression. Renal to diabetic and nondiabetic CKD in individual patients and interstitial fibrosis and protein cast formation were reduced families with one of these rare mutations. in hypertensive FHH.Add3K572 rats. Moreover, the develop- Numerous QTLs and candidate genes for CKD have been ment of proteinuria and CKD with aging was attenuated in identified in animal and GWAS.9,33 However, no variants have FHH.Add3K572 relative to FHH rats. These results support the been identified that alter the myogenic response of renal arte- view that the Q572 mutation in ADD3 contributes to the de- rioles and autoregulation of RBF. This study used novel ADD3 velopment of kidney disease, at least in FHH and MNS rats. KO and transgenic rats and a genetic complementation A remaining question is whether renal hemodynamics and approach in FHH and MNS rats to confirm that a loss-of- the susceptibility to proteinuria are altered in MNS, Wistar function mutation in ADD3 that alters ACTIN binding causes Kyoto (WKY), Buffalo, and Lewis rats that also express Q572 renal vascular dysfunction and promotes kidney disease in ADD3.28 The myogenic response of the Af-Art73–75 and the MNS and FHH rats. autoregulation of RBF76 were impaired in WKY versus SHR and MNS versus MHS rats.44 SHR and MHS rats are less sus- 9,77–79 ceptible to CKD than their control strains. Buffalo rats ACKNOWLEDGMENTS develop focal glomerular sclerosis as they age, but renal he- 9 modynamics have not been studied. We have reported that We thank Dr. Allen Cowley Jr. (Medical College of Wisconsin) for the myogenic response of the Af-Art in Lewis rats was blunted providing us with MNS and MHS rats. to the same extent as in WKY relative to SHR,75 but their susceptibility to hypertension or diabetic-induced nephropa- fi thy versus other strains has not been evaluated. These ndings DISCLOSURES suggest that the myogenic response would be reduced in other strains expressing Q572 ADD3. However, the development of Dr. Geurts reports grants from the National Institutes of Health during the proteinuria in response to hypertension or diabetes may be conduct of the study. Dr. Prokop reports grants from National Institutes of blunted relative to FHH rats because this strain has variants in Health during the conduct of the study. Dr. Roman reports grants from Na- other genes (Rab38 and Sorcs1) that impair the reuptake of tional Institutes of Health during the conduct of the study. All remaining filtered protein.10,40,80 authors have nothing to disclose. The contribution of mutations in ADD3 to the development of CKD in humans is uncertain. A region of human FUNDING chromosome 10 near ADD3 was associated with diabetic and nondiabetic CKD in black sib-pairs.81 This finding was This study was supported in part by National Institutes of Health grants replicated in a more extensive study of diabetic sib-pairs82 and AG050049 (to Dr. Fan), AG057842 (to Dr. Fan), P20GM104357 (to Dr. Ro- in a longitudinal study of whites in a Utah pedigree.83 How- man and Dr. Fan), DK104184 (to Dr. Roman) and HL138685 (to Dr. Roman) ever, the QTLs in these studies were broad (40 cM). The most and American Heart Association grant 16GRNT31200036 (to Dr. Fan). recent GWAS meta-analysis for CKD suggests that ADD3 is 5–6 Mbp from the nearest loci.33 However, our examination of SUPPLEMENTAL MATERIAL the Genome Aggregation Database (https://macarthurlab.org/ 2019/10/16/gnomad-v3-0/) revealed there are 492 nonsynon- This article contains the following supplemental material online at ymous ADD3 variants, of which 227 are damaging. A K571H http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2019080784/-/ mutation in human ADD3, analogous to the K572Q in the DCSupplemental. FHH rat, is the most prevalent variant. There are five other Supplemental Figure 1. Comparison of the expression of ADD3 variants nearby that alter charged amino acids in the actin protein in kidney of Sprague Dawley, FHH, FHH.Add3 transgenic, binding site and many others that modify phosphorylation and KO rats. sites in the tail of ADD3 that control its interactions with actin, Supplemental Figure 2. The vascular effects of the K572Q ADD3 spectrin, and membrane phospholipids. However, all of these variant are autosomal recessive. mutations are rare variants (frequency ,0.1%) that are chal- Supplemental Figure 3. Comparison of glomerular nephrin lenging to link to CKD, even in very large GWAS. In addition, staining, glomerular injury scores, percentage of renal interstitial fi- multiple isoforms of ADD3 have been identified, one of which brosis, and protein casts in hypertensive FHH and FHH.Add3 deletes amino acids 576–607 within the critical actin binding transgenic rats. region.84 Thus, we believe that additional genetic studies are needed that pool results from diabetic and hypertensive sub- jects with damaging genotypes to discern if these rare variants REFERENCES in ADD3 are associated with the CKD in more susceptible 1. Palmer ND, Ng MC, Hicks PJ, Mudgal P, Langefeld CD, Freedman BI, populations. Nevertheless, these results indicate that variants et al.: Evaluation of candidate nephropathy susceptibility genes in a in ADD3 that alter the actin cytoskeleton and impair renal genome-wide association study of African American diabetic kidney hemodynamics have the potential to increase the susceptibility disease. PLoS One 9: e88273, 2014

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JASN 31: 687–700, 2020 ADD3, vascular dysfunction and CKD 699 BASIC RESEARCH www.jasn.org

78. Stella P, Cusi D, Duzzi L, Bianchi G: Relations between hypertension to diabetic nephropathy. J Am Soc Nephrol 14[Suppl 2]: S195–S201, and glomerulosclerosis in ﬁrst-generation hybrid rats of the Milan 2003 strains. Child Nephrol Urol 11: 6–9, 1991 83. Hunt SC, Hasstedt SJ, Coon H, Camp NJ, Cawthon RM, Wu LL, et al.: 79. Brandis A, Bianchi G, Reale E, Helmchen U, Kühn K: Age-dependent Linkage of creatinine clearance to chromosome 10 in Utah pedigrees glomerulosclerosis and proteinuria occurring in rats of the Milan nor- replicates a locus for end-stage renal disease in humans and renal motensive strain and not in rats of the Milan hypertensive strain. Lab failure in the fawn-hooded rat. Kidney Int 62: 1143–1148, 2002 Invest 55: 234–243, 1986 84. Katagiri T, Ozaki K, Fujiwara T, Shimizu F, Kawai A, Okuno S, et al.: 80. Lazar J, O’Meara CC, Sarkis AB, Prisco SZ, Xu H, Fox CS, et al.: SORCS1 Cloning, expression and chromosome mapping of adducin-like 70 contributes to the development of renal disease in rats and humans. (ADDL), a human cDNA highly homologous to human erythrocyte ad- Physiol Genomics 45: 720–728, 2013 ducin. Cytogenet Cell Genet 74: 90–95, 1996 81. Freedman BI, Rich SS, Yu H, Roh BH, Bowden DW: Linkage heterogeneity of end-stage renal disease on human chromosome 10. Kidney Int 62: 770–774, 2002 82. Iyengar SK, Fox KA, Schachere M, Manzoor F, Slaughter ME, Covic AM, See related editorial, “Pulling the Hood off Genetic Susceptibility to Hyper- et al.: Linkage analysis of candidate loci for end-stage renal disease due tensive Renal Disease,” on pages 667–668.

700 JASN JASN 31: 687–700, 2020

SUPPLEMENTAL MATERIAL

SUPPLEMENTAL FIGURE LEGENDS

Supplementary Figure 1. Validation of FHH.Add3 transgenic and knockout (KO) rats.

Representative images of Western blot indicating that ADD3 (94 kDa) is expressed in the kidney of FHH.Add3 transgenic (A), SD (B), FHH.1BN congenic (C) rats that carry the reference K572 allele, but was barely detectable in FHH (A) and Add3 KO rats (C). Beta-

Actin was used as a loading control.

Supplementary Figure 2. The vascular effects of the ADD3-K572Q variant are autosomal recessive.

The middle cerebral arteries (MCA) constricted in response to the elevation of perfusion pressure in Sprague Dawley (SD) and SD.Add3 KO heterozygous (Het) rats carrying two and one of the ADD3 WT K572 alleles, respectively, but failed to constrict in SD.Add3 KO homozygous (Hom) rats. Data are presented as mean values ± SE. A two-way analysis of variance for repeated measures was used for statistical analysis, followed by a Holm-

Sidak test. P < 0.05 was considered statistically significant. * indicates significant differences compared with the corresponding values in SD and Het SD.Add3 KO rats.

Supplementary Figure 3. Comparison of the expression of glomerular nephrin staining, glomerular injury scores, renal interstitial fibrosis, and protein casts in hypertensive FHH and FHH.Add3 transgenic rats.

(A) Mean fluorescence intensities for nephrin staining per glomerulus was higher in

DOCA-salt hypertensive FHH than FHH.Add3 transgenic rats. (B) The glomerular injury score was reduced in DOCA-salt hypertensive FHH.Add3 transgenic versus FHH rats.

(C) Renal interstitial fibrosis was reduced in DOCA-salt hypertensive FHH.Add3 transgenic versus FHH rats. (D) Protein cast formation was attenuated in DOCA-salt hypertensive FHH.Add3 transgenic versus FHH rats. Data are presented as mean values

± SE. The significance of differences in mean values was determined using an unpaired t-test. * indicates a significant difference (P < 0.05) compared with the corresponding value in the control strain. Numbers in parentheses indicate the number of animals/glomeruli or areas studied.

SUPPLEMENTAL FIGURES

Supplemental Figure 1

Supplemental Figure 2

Supplemental Figure 3