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

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† ‡ † Caitlin C. O’Meara,* Michelle M. Lutz, Allison B. Sarkis,* Haiyan Xu,* Rajendra K. Kothinti,§ † † | Matthew Hoffman,* Carol Moreno,* Niloofar M. Tabatabai,§ Jozef Lazar,* † Richard J. Roman,¶ and Howard J. Jacob* **

*Human and Molecular Genetics Center and Departments of †Physiology, ‡Anesthesiology, §Medicine, |Dermatology, and **Pediatrics, Medical College of Wisconsin, Milwaukee, Wisconsin; and ¶Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, Mississippi

ABSTRACT 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.

J Am Soc Nephrol 23: 825–833, 2012. doi: 10.1681/ASN.2011080805

The majority of ESRD cases are associated with of proteinuria called Renal Failure-1 through -5 (Rf-1 diabetes, hypertension, or both; however, numer- through -5).9,10 Gene–gene interactions were found ous studies in both humans and animal models between the various Rf QTLs, because the presence suggest that renal disease susceptibility genes exist of homozygous FHH alleles in multiple Rf QTLs that are independent of the initiating factor.1–6 The resulted in a synergistic increase in proteinuria se- dissection of quantitative traits for CKD in humans verity.10 An interaction was specifically identified remains challenging due to heterogeneity and en- between Rf-1 and Rf-4, located on rat chromosome vironmental variability.7,8 Consequently, there is a 1 and 14, respectively. Van Dijk et al. generated single need to pursue other strategies for identifying genes (Rf-1a and Rf-4) and double (Rf-1a+4) congenic an- and their associated pathways that are driving CKD. imals, and found that the Rf-1a and Rf-4 single con- One solution is to use congenic rat models to in- genic animals did not show increased proteinuria vestigate regions of the rat genome that are responsible for renal impairment. One of the first examples of genetic dissection Received August 11, 2011. Accepted December 27, 2011. of renal impairment in rats was demonstrated in Published online ahead of print. Publication date available at F2 crosses between the renal disease–susceptible www.jasn.org. Fawn-hooded hypertensive (FHH) rat and the renal Correspondence: Dr.HowardJ.Jacob,MedicalCollegeof disease–resistant August Copenhagen Irish (ACI) Wisconsin, HRC5200, 8701 Watertown Plank Road, Milwaukee, rat.9,10 These studies led to the identification of five WI 53226. Email: [email protected] quantitative trait loci (QTLs) linked to the severity Copyright © 2012 by the American Society of Nephrology

J Am Soc Nephrol 23: 825–833, 2012 ISSN : 1046-6673/2305-825 825 BASIC RESEARCH www.jasn.org compared with the ACI control strain. Only the transfer of chromosome 14 from ss262968744 (15.16 Mb) to D14Hmgc18 both Rf-1a and Rf-4,asintheRf-1a+4 double congenic (19.3 Mb), a region containing 67 known and predicted genes strain, conferred a significant increase in proteinuria.11 (Supplemental Table 1). The original Rf-4 region consisted of 61.9 Mb containing As we have studied the congenics on a resistant genome 499 known and predicted genes. To narrow the region of in- background, we used a series of physiologic tools to help drive terest to begin a meaningful search for the causal variant, it is disease progression. Unilateral nephrectomy was performed necessary to physically reduce the candidate region in turn on male rats at 5 weeks of age, and animals were placed on reducing the number of candidate genes. In this study, we apurified AIN-76A rodent diet containing 0.4% NaCl and use congenic mapping to generate a minimal congenic line L-NAME–containing water after surgery. Van Dijk et al. ob- called Rf-1a+4_a, which carries only 4.1 Mbp of FHH genome served the greatest differences between Rf-1a and Rf-1a+4 in the Rf-4 region that we show significantly contributes to animals using this protocol.11 Furthermore, Rf-4 was initially proteinuria. Van Dijk et al. previously demonstrated that genes identified using an L-NAME and unilateral nephrectomy pro- in the Rf-1 region increase glomerular capillary pressure (PGC) tocol to accelerate the renal disease from 9 months to ,4 by impairing renal blood flow autoregulation.11 To explain the months.10 Animals receiving this treatment were phenotyped interaction between Rf-4 and Rf-1, they hypothesized that the for UAVat 13 weeks. The Rf-1a+4_a minimal congenic animals Rf-4 region affects integrity of the glomerular filtration barrier excreted significantly higher levels of UAV (68.6669.44 mg/d), 12 that is manifested when exposed to an increase in PGC (i.e., Rf-1). compared with Rf-1a (30.9566.07 mg/d; P=0.002) and com- Here, we assessed glomerular permeability to albumin (Palb)in pared with ACI animals (24.6066.67 mg/d; P=0.001) at 13 the Rf-1a+4_a congenics to address this hypothesis, and found weeks of age (Figure 2A). that Palb was significantly higher in Rf-1a+4_a compared with We measured BP in all strains to ensure that L-NAME control strains. treatment increased BP to a similar level in all three strains. The refined interval is only 6.6% of the original Rf-4 con- We found no significant differences in BP between strains genic region containing just 67 known and predicted genes. To (Figure 2B). initiate the search for causative variants, we analyzed the genomic sequence of the entire congenic region. Within the In Vitro Glomerular Permeability to Albumin coding sequence, we found only one benign nonsynonymous At 90 seconds after bath exchange from 6% to 4% BSA, the amino acid variant between ACI and FHH in the entire Rf-4_a fluorescent signal of the glomeruli fell to a greater percentage region, suggesting that an intergenic, intronic, or untranslated in Rf-1a and ACI animals relative to that seen in Rf-1a+4_a variant(s) is likely responsible for the Rf-4_a renal phenotype. glomeruli. The distribution of Rf-1a+4_a glomerular fluores- It has been demonstrated that conserved sequences suggest cence percentage baseline was significantly shifted to the right functionality,13–15 so we prioritized noncoding variants based compared with both ACI (P,0.001) and Rf-1a glomeruli on evolutionary conservation. Using a congenic model and (P,0.001) indicating increased permeability to albumin in comparative genomic approach, we have reduced the candi- the Rf-1a+4_a glomeruli (Figure 3A). The albumin reflection dates for the Rf-4 QTL to a handful of sequence variants. The coefficient (salb) was reduced due to an increase in glomer- results of this study are of particular interest because previous ular permeability to albumin (Palb) as calculated by 12salb studies have indicated that the homologous region on human (salb = actual change/expected [33%]). Average Palb per an- chromosome 4 is associated with various forms of CKD as imal was significantly higher in Rf-1a+4_a (0.46760.0258) indicated by both linkage16 and genome-wide association compared with ACI (0.34960.0265; P=0.009) and compared studies (GWASs).17,18 with Rf-1a (0.37360.0271; P=0.022) (Figure 3B).

Histologic Analyses RESULTS The degree of glomerular sclerosis and basement membrane thickening was determined from histologic evaluation of kid- In Vivo Phenotyping of the Rf-1a+4_a Congenic Strain ney sections stained by Gomori’s one-step trichrome stain- To narrow the Rf-4 region, a series of subcongenic lines were ing. Rf-1a+4_a animals demonstrated a significant increase created by a backcross and intercross approach (Supplemental in glomerular sclerosis (0.9460.10) compared with both the Figure 1A). Subcongenic lines were screened for the develop- Rf-1a (0.6360.06; P=0.010) and ACI (0.6360.02; P=0.011) ment of albumin excretion (UAV) and we found that animals animals as assessed by glomerular sclerosis score (Figure 3C). carrying FHH alleles from markers D14Rat78 to D14Hmgc18 Histologic analysis revealed increased glomerular, as well as demonstrated higher levels of UAV compared with other lines interstitial, fibrosis in Rf-1a+4_a kidneys compared with (Supplemental Figure 1B). To further investigate this candidate Rf-1a and ACI kidneys (Figure 3D). region, we generated a refined congenic line called Rf-1a+4_a (Figure 1). Rf-1a+4_a congenic animals are genetically identical Human Syntenic Region Analyses to the Rf-1a congenics, with the exception of the Rf-4 region in The Rf-4_a congenic region in the rat is homologous to an which 4.1 Mb of FHH genome has been integrated onto rat approximately 3.7-Mb region on human chromosome 4. The

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eGFR,18 making this an appealing renal function candidate gene. These human studies suggest that allelic variants in the region syntenic to the Rf-4 minimal congenic region could be causing kidney disease in both the human and rat. Overlap with these human loci is an important val- idation in the use of the rat to identify genetic variants that may have relevance to renal disease in humans.

Rf-4_a Sequence Annotation After alignment of reads to the reference sequence of the rat genome, 89.1% and 86.1% of nongap reference bases were covered by $3readsfortheFHHand ACI, respectively. We identified 3257 sequence variants between ACI and FHH. Of these SNPs, four were found in exons, one of which resulted in an amino acid change in the albumin gene (I431V), which was considered to be a benign amino acid substitution according to the PolyPhen prediction algorithm (http://genetics.bwh. harvard.edu/pph/), reducing the likelihood that this variant is functionally detrimen- tal to albumin function. None of the exon Figure 1. Schematic representation of the Rf-1a, Rf-1a+4,andRf-1a+4_a genotypes variants seemed to be potentially damag- on rat chromosomes 1 (RNO 1) and 14 (RNO 14) and the homologous region in the human. Black indicates FHH genotype and white indicates ACI genotype. The flanking ing, and we found no variant that might short sequence length polymorphism and SNP markers for the Rf-1a, Rf-4 and Rf-4_a cause mis-splicing of the exon. To prior- congenic regions are shown to the left of the rat chromosome, and the position in itize noncoding sequence variants, we Mbp of each marker is shown to the right. The flanking markers for the Rf-4_a region assessed species conservation to help pre- are shown in bold. The homologous region in the human is located on chromosome 4 dict functionality of variants within in- from approximately 74.3 to 78 Mb, and the orientation of this region in human is tronic or intergenic regions. We identified inverted compared with that of the rat. (¥) indicates human QTL for GFR,16 and (Δ)and 15 variants with a high conservation score (§) indicate GWAS loci for GFR17 and serum magnesium,18 respectively. (.0.75) based on the phasCons algorithm (Table 1).14 Only one highly conserved intergenic orientation of this region is inversed in the rat compared with variant found in the FHH strain, at position 17,821,228 of the human, indicating a chromosomal rearrangement be- chromosome 14, was completely conserved between eight of tween the human and rodent. the nine annotated species (there was no alignment for cow at A Framingham study by Fox et al. mapped QTLs for both this position), including ACI and Brown Norway. This suggests GFR and creatinine clearance to human chromosome 4, that this conserved allele is under evolutionary selection and, within the human homologous region to the Rf-4 QTL.16 variation may be important to physiologic function. This highly The peak of this QTL was nearest to marker D4S2367,and conserved variant was predicted by TFSearch (http://www.cbrc. the estimated span overlaps the human genome homologous jp/research/db/TFSEARCH.html) to cause a loss of Nrf2 tran- to the Rf-4 minimal congenic region. Furthermore, a 2009 scription factor binding site in FHH. To test the validity of this GWAS identified a locus significantly associated with GFR prediction, we performed electrophoretic mobility shift assay estimated by serum creatinine (eGFRcreat) on human chro- (EMSA) with nuclear proteins isolated from Brown Norway mosome 4.17 The most significant single nucleotide polymor- rat kidney and respective oligonucleotide probes from ACI and phism (SNP) in this region, rs17319721, is located within an FHH. We found that nuclear protein binding to the ACI se- intron of the SHROOM3 gene, which is 1 of the 67 candidate quence is significantly higher compared with the FHH sequence genes in the Rf-4 minimal congenic region. SHROOM3 was in vitro (P,0.001). Furthermore, supershift assays using Nrf2 also identified in a separate GWAS as being linked to serum antibody supported that renal Nrf2 can bind to the ACI se- magnesium concentration and kidney function as assessed by quence, but the FHH variant nucleotide adversely affects

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development of proteinuria only in the presence of Rf-1a.11 The Rf-1a+4 double congenic rat allowed us reduce the size of the critical interval of Rf-4 from 61.9 Mb to just 4.1 Mbp. This interval, called Rf-4_a,is just 6.6% of the original Rf-4 region, reducing the candidate gene list to just 67 known and predicted genes. Indeed, Rf-1a+4_a congenic animals excreted significantly higher levels of albumin than Rf-1a congenics, and this increased albuminuria was associated with an increase in glomerular permeability to albumin, indicating damage to the glomerular filtration barrier. Figure 2. Rf-1a+4_a animals excrete higher levels of albumin compared with ACI and The long-term goals of this renal failure “ ” Rf-1a, but BP is not different between strains. (A) The susceptibility loci are on a re- ( Rf ) project have been to identify the sistant genome background (ACI), and therefore animals phenotyped for albumin underlying causal variants and to study excretion were unilaterally nephrectomized at 5 weeks of age and hypertension was the nature of the Rf QTL interactions. It is induced by L-NAME diluted in the drinking water. Animals were placed into metabolic known that albuminuria can occur as a re- cages 8 weeks after unilateral nephrectomy, and 24-hour samples were analyzed for sult of changes in renal hemodynamics and albumin excretion. Albuminuria is significantly elevated in Rf-1a+4_a compared with resultant elevations in glomerular capillary Rf-1 and ACI. n=7, n=10, and n=10 animals for ACI, Rf-1, and Rf-1a+4_a, respectively. pressure (P ), leading to increases in GFR # # GC **P 0.01. ***P 0.001. (B) MAP is not different in Rf-1a+4_a animals compared with and the filtered load of protein, changes in ACI and Rf-1a. BP was recorded in 14-week-old awake animals by radiotelemetry after the glomerular filtration barrier to pro- the metabolic cage experiment. n=9, n=12, and n=10 animals for ACI, Rf-1a,and teins, and/or altered protein trafficking Rf-1a+4_a, respectively. and reuptake of filtered proteins in the proximal tubule.21,22 Van Dijk et al. pro- binding of Nrf2 to this region (Supplemental Figure 2). These posed that the Rf-4 region affects the integrity of the glomer- data demonstrate that intergenic variants can affect transcrip- ular filtration barrier that is manifested when exposed to an tion factor binding in Rf-1a+4_a animals; therefore, tran- increase in PGC (Rf-1), particularly in the presence of stressors scriptional regulation of gene expression could contribute to such as L-NAME and reduced renal mass.11 To address this the Rf-4_a renal phenotype. hypothesis, we measured Palb in glomeruli isolated from 12- to Kidney RNA from 6-week-old animals was analyzed for 13-week-old rats, before significant development of proteinuria, expression of genes up- or downstream of the Nrf2 binding and found that indeed the in vitro permeability to albumin was site variant as well as known genes in the entire Rf-4_a region. significantly higher in Rf-1a+4_a compared with Rf-1a and ACI, Fold change of Rf-1a and Rf-1a+4_a gene expression was com- supporting the hypothesis that an insult at the glomerulus in pared with ACI (Supplemental Table 2). We detected expres- combination with changes in PGC are required for the presence sion of all known genes except for Ereg and Epgn in the kidney, of measureable proteinuria in this model. This is the first in- but found no genes within the congenic region to be signifi- stance in which we have identified a mechanism of renal im- cantly differentially expressed between strains. pairment in two separate Rf QTL, and demonstrated that the interaction between these two mechanisms is required to produce a proteinuria phenotype. DISCUSSION We found greater heterogeneity in the measurement of in vitro glomerular permeability in Rf-1a+4_a compared with Of the five Rf- QTLs mapped in crosses between ACI and FHH, Rf-1a and ACI. Increased heterogeneity would be expected our group has previously refined three of them (Rf-1, Rf-2,and given the focal nature of segmental glomerular sclerosis in Rf-3)19,20 using a congenic strategy, and we have successfully iden- FHH rats.23–25 That is, the barrier function of all glomeruli tified causative genes underlying Rf-1 (J.LazarandH.J.Jacob, is not equally affected; only isolated regions of a kidney display 2011, unpublished data) and Rf-2.20 In this study, we utilized a impaired (eventually sclerosed) glomeruli, whereas glomeruli similar approach to greatly refine the Rf-4 QTL as well. We reduced in other regions of the kidney appear healthy. Because of this this region to a small enough interval in which we can initiate a heterogeneity, it was necessary to assess a larger population of feasible search for causative variants by sequence annotation. glomeruli from each strain to improve the sensitivity of the Combining the Rf-1 and Rf-4 QTLs in the double congenic assay. By using change in fluorescence as a high throughput model was crucial for successfully refining the Rf-4 region. measurement of change in volume (DV),wewereabletoas- Previous studies showed that Rf-4 contributes to the sess on average approximately 21 glomeruli per animal, about

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cell motility.26 Actin binding is known to be important in maintaining the glomerular filtration barrier integrity,27,28 making Shroom3 an interesting candidate for glomerular permeability in the Rf-4_a region. However, we did not find amino acid changes between ACI and FHH, suggesting that this gene is not segregating with the phenotypic differences between ACI and FHH mapped to this QTL. We found only four coding variants within the entire Rf-4_a region, and only one of these resulted in a nonsynonymous, benign amino acid change. Few of these coding variants were conserved among species, and none were predicted to be damaging to protein function. Therefore, the Rf-4_a causative variant(s) is likely not coding, but rather an intergenic or intronic regulatory element. Some GWASs have Figure 3. Rf-1a+4_a animals have increased glomerular permeability to albumin, and shown significant associations of inter- a higher percentage of glomerular sclerosis compared with ACI and Rf-1a. (A) Animals genic regions with disease,13,29,30 indicat- were infused with high molecular mass (250 kD) FITC-labeled dextran 3 minutes ing that nongenic sequences can also fi before sacri ce to allow perfusion of glomerular capillaries. Glomeruli were isolated influence a complex disease. Noncoding in 6% BSA and total fluorescence was measured. Media were switched to 4% BSA sequence accounts for a vast majority of and the change in fluorescence over time was measured. Distribution of glomerular swelling, as determined by decrease in fluorescence at 90 seconds after bath ex- sequence variants found in mammalian ge- change from 6% to 4% BSA compared with baseline fluorescence of Rf-1a+4_a (green nomes; however, determining functionality 14 line) glomeruli is shifted to the right compared with Rf-1 (red line) and ACI (black line), of these sequences remains a challenge. indicating a significant increase in glomerular permeability. # and § indicate P,0.001 Comparative sequence analysis has been versus ACI and Rf-1a, respectively. n=117, n=144, and n=158 glomeruli measured for one strategy to identify nongenic sequences each group for ACI, Rf-1a,andRf-1a+4_a respectively. (B) Glomerular permeability that have regulatory function.31 On the per animal was higher on average for Rf-1a+4_a compared with ACI and Rf-1a. basis of prioritizing intergenic variants by 2 s s Palb (1 alb, where alb = actual change/expected [33%]) was calculated for each conservation, we preliminarily narrowed glomeruli, and the mean of means for each group is represented. n=6, n=8, and n=6 the list of likely causative variants to just animals for ACI, Rf-1a,andRf-1a+4_a, respectively. (C) Rf-1a+4_a kidneys have in- 15 in the Rf-4_a region. creased presence of glomerular sclerosis compared with ACI and Rf-1a at 15 weeks of The Rf-4_a region carries a variant, age. Thirty glomeruli from four kidneys for each strain were scored for percentage of glomerular sclerosis using a scoring scale from 0 (no sclerosis) to 4 (complete scle- which is otherwise most highly conserved, rosis). (D) ACI and Rf-1a kidneys present little to no glomerular sclerosis and interstitial and is predicted to cause the loss of an Nrf2 fibrosis (panels 1 and 2), whereas Rf-1a+4_a kidneys showed an increased abundance transcription factor binding site. Activa- of glomerular sclerosis (+) as well as interstitial fibrosis (→) (panel 3). Bar indicates tion of Nrf2, a master regulator of antiox- 50 mM. *P#0.05. **P#0.01. idant molecules, plays a protective role against renal disease progression.32,33 Spe- cifically, Nrf2-deficient mice demonstrate four times the number of glomeruli assessed using the tradi- increased glomerular sclerosis in response to hyperglyce- 20 34 tional in vitro Palb methods. Mean arterial pressure (MAP) is mia, whereas elevated Nrf2 levels by using a pharmacological notelevatedintheRf-1a+4_a congenics, further supporting agent inducer improve renal function in mice.35 Thus, de- the hypothesis that genes in this region are directly affecting creased binding affinity of Nrf2 to the regulatory region of the kidney, and the observed UAV is not secondary to hyper- yet to be identified gene(s) in Rf-1a+4_a animals could ad- tension. versely influence the antioxidant response and thereby con- The Rf-4_a syntenic region has been associated with mul- tribute to increased glomerular damage. Although we found tiple renal function loci in humans. Two separate GWASs have no gene expression differences in kidneys of young (6-week- linked the SHROOM3 gene to renal function,17,18 indicating that old) animals, the role of Nrf2, either alone or in association certain human alleles in, or around, this gene confer suscep- with other regulatory elements, could be influencing gene ex- tibility to renal function. Shroom3 encodes an F-actin binding pression only after the induction of oxidative stress by unilat- protein that has been shown to play a role in epithelium-like eral nephrectomy and L-NAME treatments, because these

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Table 1. Variants between ACI and FHH in the Rf-4_a region that are highly Generation of the Rf-1a+4_a Strain conserved between species (conservation score .0.75) The congenic breeding strategy is outlined in Conservation Supplemental Figure 1A. The development of Position Start Stop ACI FHH Score the Rf-1a+4_a minimal congenic strain was ini- Intergenic 15,168,618 15,168,618 C G 0.96 tiated by crossing Rf-1a (ACI.FHH-[D1Rat74- Intergenic 15,168,719 15,168,719 C G 0.99 D1Rat90]) single congenic males to Rf-1a+4 Intergenic 15,168,744 15,168,744 C A 0.99 (ACI.FHH-[D1Mit18-D1Rat90]/[D14Mit11- Intergenic 15,228,978 15,228,978 A C 1.00 D14Rat33/D14Rat65-D14Rat90]) double con- Intergenic 15,240,810 15,240,810 G C 0.89 genicfemales.Finemapgenotypinginour Intergenic 15,247,380 15,247,380 TA T 0.85 hands revealed that the Rf-1a congenic region Intergenic 16,411,415 16,411,415 AT A 0.97 is identical in both Rf-1a and Rf-1a+4 strains, Intergenic 17,765,698 17,765,698 A G 0.91 spanning from D1Mit18 to D1Rat90; therefore, Intergenic 17,816,838 17,816,838 T A 0.92 F1 generation animals were heterozygous for Intergenic 17,821,228 17,821,228 C T 1.00 the Rf-4 QTL but remained FHH homozygous Intergenic 17,835,462 17,835,462 G T 0.96 in the Rf-1a region. F1 animals were inter- Intergenic 17,883,002 17,883,002 A T 0.85 Intergenic 18,734,287 18,734,287 A G 1.00 crossed and F2 generation offspring were geno- fl Exon (Afp: D282D) 19,101,441 19,101,441 A G 0.99 typed using a previously described uorescent Intergenic 19,247,351 19,247,351 C T 1.00 genotyping protocol to identify animals with a Start and stop are the base positions of the variant on rat chromosome 14. The position column in- desirable recombination within the Rf-4 re- dicates if the variant is intergenic, intronic, or exonic (followed by the gene symbol and amino acid gion.36 Animals that had inherited a recombi- position if applicable). nation of interest were then backcrossed to the Rf-1a congenic line, and offspring carrying the Rf-4 recombined congenic interval were inter- stressors are required for the manifestation of the Rf-4 phe- crossed to fix the recombinant region to homozygosity. Subcongenic notype. Further expression analysis at different ages and stages animals were phenotyped for UAV to map the genetic region con- of proteinuria could demonstrate differences among our tributing to the renal impairment phenotype, and we found that FHH strains. Additional studies of transcription factor activity in genotype in the region between approximately D14Rat78 (17.6 Mbp) our congenic strains are also required to resolve the precise and D14Hmgc18 (19.3 Mbp) caused increased UAV(Supplemental Fig- mechanism of renal damage in the Rf-4_a interval. ure 1B). To investigate this small region of the genome, we generated a Many GWAS SNPs are noncoding variants, and therefore minimal congenic line called Rf-1a+4_a (ACI.FHH-[D1Mit18- few GWAS have pinpointed the causative element underlying D1Rat90]/[D14Rat98-D14Hmgc18]/Mcwi) (RGD ID 4145374). Weuti- loci. Rf-1a+4_a may serve as a useful model to provide insight lized SNP genotyping to fine map the upper boundary of Rf-4_a,and into the characterization of noncoding sequence variants that found that this region extends to ss262968744, located at 15.16 Mbp. affect a complex disease, and these sequences can be compared across species to help identify causative variation in human Phenotyping for Albuminuria and BP forms of renal disease. In this study, we characterized a mech- Male rats aged 5–6 weeks were anesthetized with a cocktail of keta- anism of proteinuria in the Rf-4_a congenic region. This 4.1-Mb mine (30 mg/kg), xylazine (2.5 mg/kg), and acepromazine (0.6 mg/kg). interval is homologous to human loci and QTL that have been The right kidney was exposed by a retroperitoneal incision, the implicated in renal function, supporting the need to further renal artery and vein were ligated, and the kidney was removed. investigate this region of the genome. It is likely that a variant(s) After surgery, 150 mg/L of L-NAME (Sigma-Aldrich, St. Louis, MO) in noncoding sequence is responsible for the Rf-4_a pheno- was added to the rats’ drinking water and their chow switched from type, making the Rf-4_a a useful model for studying the phys- Laboratory Rodent Diet 5001 (PMI Nutrition International Inc, iologic effect of nongenic sequences in renal disease. Additional Brentwood, MO) to a purified AIN-76A rodent diet containing fi studies will be required to prove the speci c variant(s) respon- 0.4% NaCl (Dyets Inc, Bethlehem, PA). This chow and L-NAME sible for increased glomerular permeability and the molecular dose were maintained ad libitum throughout the remainder of the mechanisms causing the renal impairment phenotype in the protocol. Eight weeks after unilateral nephrectomy, the rats were Rf-1a+4_a animals. housed in metabolic cages (Nalgene, Rochester, NY) for urine collection. Rats were acclimated to metabolic cages for 2 days, and urine CONCISE METHODS was then collected for two consecutive 24-hour periods and albumin concentration was determined using the Albumin Blue 580 assay Animal Care (Molecular Probes, Eugene, Oregon).37,38 The rats were housed in the Biomedical Resource Center of the Med- BP was measured 9 weeks after unilateral nephrectomy, imme- ical College of Wisconsin, an American Association for the Accred- diately after the final urine collection. MAP was measured in awake itation of Laboratory Animal Care–approved facility. The local Animal rats by radiotelemetry (Data Sciences Inc, St. Paul, MN) as described Care and Use Committee approved all protocols used in these studies. previously.39 Telemetry transmitters (TA11PA-C40) were implanted

830 Journal of the American Society of Nephrology J Am Soc Nephrol 23: 825–833, 2012 www.jasn.org BASIC RESEARCH subcutaneously (under isoflurane anesthesia), and the catheter was For DNA probe, high-performance liquid chromatography purified inserted into the abdominal aorta via the femoral artery. Animals oligonucleotides with forward and reverse sequences of Rf-4_a var- were allowed 4 days for recovery after surgery, and BP was then re- iant region of FHH and the respective region of ACI were synthesized corded at 500 Hz in conscious, freely moving animals for 3 consec- by Integrated DNA Technologies (IDT, Coralville, IA). The ACI oli- utive days. Ten-second intervals were continuously recorded every gonucleotide had the forward sequence of 59-TGGGTGACTTTGTA- two minutes, and these data were averaged over a 3-hour period each GACTCTTCCGGTTTTCCGTGGTA-39,whereasC at position 19 day to estimate MAP. was replaced with a T in the FHH. Respective forward and reverse oligonucleotides were annealed and the double-stranded DNA was In Vitro Fluorescent Glomerular Permeability Assay labeled with DIG-11-ddUTP using a recombinant Terminal Trans- Male animals aged 12–13 weeks were anesthetized with isofluorine ferase (20 U/ml) (DIG Gel Shift Kit, Second Generation; Roche, In- and infused with a bolus of high molecular mass (250 kD) FITC- dianapolis, IN). Binding assays were performed with 5 mg of nuclear labeled dextran (Sigma-Aldrich) dissolved in 0.7 mL of saline at a proteins and 0.08 pmol of each DIG-labeled probe. For the supershift dose of 75 mg/kg body wt via femoral vein catheterization. After 3 assay, binding was performed with nuclear proteins incubated with minutes of equilibration, animals were sacrificed, both kidneys were 4 mg of Nrf2 (C-20, sc-722 X) antibody (Santa Cruz Biotechnology, removed, and the glomeruli were isolated using a differential sieving Santa Cruz, CA). After gel electrophoresis and blotting onto mem- technique as described previously.40 The in vitro glomerular perme- brane, chemiluminescence detection and x-ray film exposure were ability assay was adapted from a previously described method.20,40 performed, and band intensities were measured with ImageJ software Savin et al. determined DV by measuring glomerular diameter, (National Institutes of Health). whereas we directly assessed DV by dilution of a fluorescent volume fl marker (250 kD-FITC dextran). The total uorescent intensity of RNA Extraction and Quantitative PCR each glomerulus was measured and recorded in real time (1 obser- Six-week-old animals were sacrificed and whole kidneys were re- vation/sec) using the Incyte1 program. After baseline recording, the moved and placed into RNAlater (Ambion Life Technologies, Grand perfusion media were switched from 6% BSA to 4% BSA to expose Island, NY). Total RNA was extracted from whole kidneys with fl glomeruli to a hypo-oncotic environment. The uorescent level 90 Trizol reagent (Invitrogen) and cDNA was synthesized using Super- seconds after bath exchange to the 4% BSA was used to calculate the script III reverse transcription (Invitrogen) according to the man- fl fi s albumin re ection coef cient ( alb) and Palb. The measured change ufacturer’s instructions. Quantitative PCR was performed by using s in volume/expected change (33%) determined the alb, and Palb was the QuantiTect SYBR Green PCR kit (Qiagen, Valencia, CA) and calculated by 1 2 salb. the Rotorgene 3000. Cycling was performed at 95°C for 15 minutes, 94°C for 20 seconds, 60°C for 20 seconds, 70°C for 20 seconds for Glomerular Histology 40 cycles, and 70°C for 5 minutes. mRNA expression was normalized After the metabolic cage experiment, 14-week-old unilaterally ne- to b-actin as an endogenous control, and relative expression was fi (-DDCT) phrectomized animals were sacri ced and the left kidney was removed calculated by 2 method versus ACI. and immediately placed in 10% buffered formalin (Sigma-Aldrich) for fixation. Fixed kidneys were sectioned and stained using Gomori’s Statistical Analyses one-step trichrome stain for histologic analysis. Individual glomeruli Data are presented as means 6 SEM. Albuminuria, glomerular index were scored on a scale of a 0 through 4, with 0 indicating no damage, 2 score, and average Palb data were analyzed using ANOVA, followed representing loss of 50% of glomerular capillary area, and 4 indicat- by a Holm–Sidak multiple comparison test using Sigma Plot 11.0 ing complete loss of the glomerular filtration area. software. The BP data set was not normally distributed and was analyzed by a Kruskal–Wallis one-way ANOVA on ranks. Difference in Congenic Region Sequence Analyses distribution of fluorescent dilution of FITC dextran between the ACI and FHH genomic DNA was sequenced on an Illumina three strains was analyzed using standard SAS statistical software ’ HiSEquation 2000 according to the manufacturer s instructions (Il- package (version 9.2; SAS Institute, Cary, NC). Kolmogorov–Smirnov ’ lumina, San Diego, CA). We used Illumina sCASAVAsoftwareto nonparametric tests were used for normality tests and two sample align the paired-end reads to the reference genome and identify var- comparisons. iants. Variants with a read depth .3 and occurring in .50% of the reads were annotated with ANNOVAR software.41 Sequence comparisons were made between the ACI and FHH within the minimal congenic region. ACKNOWLEDGMENTS

fi EMSA This study was performed with nancial support from the National Nuclear protein preparation and EMSA were performed as described Heart, Lung, and Blood Institute (NHLBI-5R01HL069321) to H.J.J. previously.42 Briefly, kidney was isolated from male Brown Norway rat, homogenized in PBS, and protein extracts were prepared in the absence of EDTA and quantified. Expression of Nrf2 in the nuclear DISCLOSURES fraction was confirmed by Western blot analysis (data not shown). None.

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