BASIC RESEARCH www.jasn.org

Genetic Susceptibility and Loss of Nr4a1 EnhancesMacrophage-Mediated Renal Injury in CKD

Lindsey Westbrook,* Ashley C. Johnson,* Kevin R. Regner,† Jan M. Williams,*‡

David L. Mattson,§ Patrick B. Kyle,| Jeffery R. Henegar,| and Michael R. Garrett*‡

Departments of *Pharmacology and Toxicology, ‡Medicine, and |Pathology, University of Mississippi Medical Center,Jackson, Mississippi; and Departments of †Medicine and §Physiology, Medical College of Wisconsin, Milwaukee,Wisconsin

ABSTRACTNuclear hormone receptors of the NR4A subgroup have been implicated in cancer, atherosclerosis, andmetabolic disease. However, little is known about the role of these receptors in kidney health or disease.Nr4a1-deficient rats (Nr4a12/2) developed on a genetic background susceptible to kidney injury (fawn-hooded hypertensive rat [FHH]) were evaluated for BP, proteinuria, renal function, and metabolic param-eters from 4 to 24 weeks-of-age. By week 24, Nr4a12/2 rats exhibited significantly higher proteinuria(approximately 4-fold) and decreased GFR compared with FHH controls. The severity of tubular atrophy,tubular casts, and interstitial fibrosis increased significantly in Nr4a12/2 rats and was accompanied by alarge increase in immune cell infiltration, predominantly macrophages and to a lesser extent T cells and Bcells. Global transcriptome and network analyses at weeks 8, 16, and 24 identified several proinflamma-tory genes and pathways differentially regulated between strains. Bone marrow crosstransplantationstudies demonstrated that kidney injury inNr4a12/2 rats was almost completely rescued by bonemarrowtransplanted from FHH controls. In vitro, macrophages isolated from Nr4a12/2 rats demonstrated in-creased immune activation compared with FHH-derived macrophages. In summary, the loss of Nr4a1 inimmune cells appears to cause the increased kidney injury and reduced renal function observed in theNr4a12/2 model.

J Am Soc Nephrol 25: 2499–2510, 2014. doi: 10.1681/ASN.2013070786

The incidence of CKD has increased over the last 2decades.1 Current estimates suggest that approxi-mately 10% of adults in the United States haveCKD.2 Considering only federal Medicare spend-ing, the direct economic effect of CKD is staggering.The cost to treat CKD (predialysis) in 2010 was $41billion, in addition to ESRD (dialysis and trans-plantation) costs estimated at $32.9 billion.3 A sig-nificant problem is that current treatment optionsare not ideal and only serve to slow the progressionof CKD. Thus, the identification of genes and/orbiologic pathways that mediate progressive kidneydisease is critical to both developing new and/orimproved treatments for CKD and curbing the as-sociated financial burden.

Nuclear hormone receptors are transcriptionfactors that play a role in a variety of biologicprocesses and are induced in multiple tissues bydiverse stimuli, such as b-adrenoceptor agonists,

cold, fatty acids, glucose, insulin, and cholesterol.4

The nuclear receptor (NR) 4A subgroup of nuclearhormone receptors is composed of three members:Nr4a1 (Nur77), Nr4a2 (Nurr1), and Nr4a3 (Nor1).In contrast with other NRs, the activity of NR4Areceptor family is ligand independent and regulatedat the level of gene expression and protein stability.5

The NR4A subgroup has been linked to cancer,6

Received July 25, 2013. Accepted February 5, 2014.

L.W. and A.C.J. contributed equally to this work.

Published online ahead of print. Publication date available atwww.jasn.org.

Correspondence: Dr. Michael R. Garrett, Department of Phar-macology and Toxicology, University of Mississippi MedicalCenter, 2500 North State Street, Jackson, MS 39216. Email:mrgarrett@umc.edu

Copyright © 2014 by the American Society of Nephrology

J Am Soc Nephrol 25: 2499–2510, 2014 ISSN : 1046-6673/2511-2499 2499

obesity,7 diabetes,8 and vascular disease.9 A significant role forNR4A1 has been established in chronic inflammatory diseasessuch as atherosclerosis, psoriasis, and rheumatoid arthritis.10

Although inflammation is a key factor in the pathogenesis of avariety of kidney diseases, little is known about the role thatNR4A receptors may play in kidney health or disease.

This study aimed to explore the role of Nr4a1 in the contextof a geneticmodel (fawn-hooded hypertensive [FHH] rat) thatis sensitive to cardiovascular and kidney disease. Weperformed a longitudinal studyof BP, renal injury, proteinuria,and renal function in Nr4a12/2 and FHH control animalsfrom 4 to 24 weeks-of-age. BP between strains was similar,but kidneys from Nr4a12/2 rats demonstrated prominent tu-bulointerstitial injury and inflammation, suggesting that themechanism of injury was immune mediated. Bone marrowcrosstransplantation and in vitro studies using macrophagesisolated from both strains suggest that loss of Nr4a1 in im-mune cells such as macrophages is the likely cause of kidneyinjury and renal dysfunction in Nr4a12/2 rats. In summary,these findings suggest a novel and previously unknown role forNr4a1 in the pathogenesis of kidney disease.

RESULTS

Association of Nr4a1 Mutation with Proteinuria byLinkage AnalysisTo confirm and validate that the point mutation (TAC [tyro-sine] to TAA [stop]) in Nr4a12/2 animals was associated withproteinuria (renal injury), a genetic analysis was performedusing an F2 (FHH3Nr4a12 /2) segregating population(n=175 animals) (Supplemental Figure 1). Several parametersincluding body weight, kidney weight, proteinuria, fluid in-take, and urine output were measured at 8 and 12 weeks-of-age. At both time points, animals with the A/A genotype(which results in a premature stop codon) exhibited signifi-cantly higher proteinuria compared with the wild-type C/Cgenotype (Supplemental Figure 1). The linkage analysis alsodemonstrated that animals having the A/A genotype were sig-nificantly smaller (23362.1 g), while having significantlylarger kidneys (10.560.1 mg/g body wt) compared with thewild-type G/G genotype (24962.1 g and 9.560.1 mg/g bodywt, respectively; P,0.01). In summary, linkage analysis pro-vided conclusive evidence that the Nr4a1 locus (point muta-tion) is linked to increased proteinuria and associated renalmeasures.

Temporal Changes in Renal, Cardiovascular, andMetabolic TraitsFHH and Nr4a12/2 rats were evaluated for changes in BP,proteinuria, and renal function from 4 to 24 weeks-of-age(Figure 1, A and B). Consistent with the linkage analysis,Nr4a12/2 rats demonstrated significantly higher albuminuriaand proteinuria compared with FHH rats starting at week 8(Figure 1B, Table 1). By week 24, Nr4a12/2 rats exhibited

approximately 3-fold higher proteinuria compared with con-trols. A similar increase in telemetry-measured BP was ob-served from weeks 8 to 20 between strains. BP measured atweek 24 (by indwelling catheter) also demonstrated similar BPbetween strains (Figure 1B). Likewise, no significant differ-ence in cardiac hypertrophy was observed throughout thetime course (Table 1). Renal hemodynamics were evaluatedat weeks 16 and 24 to more comprehensively evaluate renalfunction. No significant difference in renal blood flow (RBF)or GFR was observed between strains at week 16 (Figure 1C).By week 24, a significant reduction in GFR was observed inNr4a12/2 rats compared with FHH rats, which was not accom-panied by changes in BP. As expected, impairment of RBFautoregulation was observed in the FHH rats because RBF in-creased when renal perfusion pressure varied from 90 to 140mmHg (Supplemental Figure 2). A similar impairment in RBFautoregulation was also observed in the Nr4a12/2 rats. Signifi-cant increases in kidney weight and BUN and a significantdecrease in creatinine clearance was observed in Nr4a12/2

(compared with FHH) at week 8, 16, and 24 (Table 1). Severalother metabolic parameters, including elevated cholesterol andtriglycerides was observed in the Nr4a12/2 as well as significantlipid deposits in the liver (Table 1, Supplemental Figure 3).

Characterization of Glomerular, Tubular, and VascularRenal InjuryThe severity of kidney injury in FHH and Nr4a12/2 rats wasassessed at weeks 8, 16, and 24. Moderate increases in mesangialmatrix expansion and glomerulosclerosis were observed inboth stains with progressive injury from weeks 8 to 24. How-ever, there was no significant difference in the severity of glo-merular injury between strains (Figure 2, A and B). Electronmicroscopy analysis showed that glomeruli exhibited a similardegree of mesangial expansion and podocyte foot processeffacement between strains (Supplemental Figure 4). The severityof tubular injury by evaluation of tubular atrophy, dilation,and protein casts was significantly increased fromweeks 16 to24 in Nr4a12/2 rats compared with FHH rats (SupplementalFigure 5). Consistent with tubular injury, the degree of in-terstitial fibrosis was significantly increased in Nr4a12/2 rats(approximately 2-fold) compared with controls starting atweek 16 (Figure 2, A and B). Electron microscopy analysisdemonstrated enhanced matrix formation between adjacenttubules and cell degeneration in proximal tubules cells ofNr4a12/2 rats (Supplemental Figure 4). No difference in renalarteriole morphology was detected between strains from 8 to 24weeks-of-age.

Increased macrophage infiltration was observed in kidneyfromNr4a12/2 rats starting at week 16 in the tubulointerstitialregion and by week 24 was 3- to 4-fold higher than FHH rats(Figure 2D). No significant difference in glomerular macro-phage infiltration was observed until week 24. Kidneys fromNr4a12/2 rats also demonstrated a significant increase in T- andB-cell infiltration by week 24 (CD43; 34.267.4 versus 16.362.2foci per320 field; CD22: 9.362.04 versus 4.461.2, respectively;

2500 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 2499–2510, 2014

BASIC RESEARCH www.jasn.org

P,0.05), but to a lesser extent compared with macrophageinfiltration. Gene expression markers of macrophages (CD68and SOCS3) and tubular injury genes (Kim-1 and Ngal) wereupregulated in the Nr4a12/2 rats (Supplemental Figure 5).Taken together, these findings indicate that loss of Nr4a1exacerbates macrophage infiltration predominantly in the tubu-lointerstitial region and culminates in significant injury and renalfunction decline.

Time Course Transcriptome AnalysesGene expression profiling was performed at weeks 8, 16, and 24using kidney from FHH andNr4a12/2 rats. A two-way ANOVA

was performed to identify genes that were significantly differentbetween strains (Nr4a12/2 and FHH) and over time (weeks 8,16, and 240; i.e., strain [n=277], time [n=975], and interaction[n=635]) using fold change $61.2 and a P,0.01. Principalcomponent analysis identified distinct differences between sam-ple groups and gene expression changes over time. At week 8,gene expression patterns were similar between Nr4a12/2 andFHH kidneys. However, at weeks 16–24, Nr4a12/2 gene expres-sion differences were distinct from FHH rats and each other(Supplemental Figure 6), whereas FHH gene expression differ-ences were similar from weeks 16 and 24. Ingenuity PathwayAnalysis identified inflammatory response as the top biologic

Figure 1. Temporal evaluation of BP, proteinuria, and renal hemodynamics in FHH and Nr4a12/2 rats. (A) Proteinuria and telemetry-measured BP are assessed in the FHH and Nr4a12/2 rats from 4 to 20 weeks-of-age (n=28 per group at week 4 to n=6 at week 20; 6 ratsare euthanized at weeks 8, 12, and 16). (B) Proteinuria and MAP at week 24 (n=6–8 per group; separate group from A). BP is measuredin conscious animals by indwelling animals. (C) RBF, GFR, and MAP at weeks 16 and 24 for animals (n=6–8 per group). *P,0.05compared with FHH (same time point). Mean values6SEM are presented. SBP, systolic BP; BW, body weight; MAP, mean arterialpressure; KW, kidney weight.

J Am Soc Nephrol 25: 2499–2510, 2014 Loss of Nr4a1 and Kidney Injury 2501

www.jasn.org BASIC RESEARCH

function associated with gene expression differences. Identifiedgenes were associated with key upstream regulators of inflam-mation (e.g., LPS, IFN-ɣ, IL-1b, IL-6, and TNF-a). Importantinflammatory genes observed to be differentially expressed (bymicroarray) betweenNr4a12/2 and FHHwere validated by real-time PCR, including Ccr2, Tgf-b1, Tgf-b2, Ifn-ɣ, Tnf-a, Il-1b,and Il-6 (Supplemental Figure 5).

Bone Marrow Cross-Transplantation Studies betweenNr4a12/2 and FHH RatsOn the basis of the specificity of the histologic injury, increasein immune cell infiltration, and inflammatory gene expressiondifferences in kidney from Nr4a12/2 rats (compared withFHH rats), we aimed to determine whether loss of Nr4a1 pro-motes kidney injury via a mechanism involving immune cells.Bone marrow (bm) crosstransplantation studies were per-formed in three groups of irradiated (irr) animals: (1) FHHbm

into FHHirr (FHH control), (2) FHHbm into Nr4a1irr2/2

(experimental), and (3) Nr4a1bm2/2 into Nr4a1irr

2/2

(Nr4a12/2 control). The transplant of bone marrow fromFHH into Nr4a1 was confirmed by the identification of FHHgenotype in Nr4a1 animals (Supplemental Figure 7). FHHbm

into Nr4a1irr2/2 animals showed a significant attenuation of

proteinuria starting as early as week 12 (4 weeks after trans-plantation) and continued through week 24 compared withthe Nr4a1bm

2/2 into Nr4a1irr2/2 (control) animals (Figure

3A). Creatine clearance was similar between FHH controlscompared with FHHbm into Nr4a1irr

2/2 animals, but bothwere significantly higher compared with Nr4a12/2 controlanimals (Figure 3B). Improved renal injury and renal functionin FHHbm into Nr4a1irr

2/2 animals was corroborated byreduced tubular injury (Supplemental Figure 7), interstitialfibrosis, and tubulointerstitialmacrophage infiltration (Figure 3,C and D) compared with Nr4a12/2 controls. FHHbm intoNr4a1irr

2/2 animals demonstrated a significant reduction ofserum cholesterol and triglycerides compared with Nr4a12/2

controls (Supplemental Figure 7).

Thioglycolate-Elicited and Bone Marrow–DerivedMacrophage Isolation and Cell CultureThioglycolate-elicited immune cells were isolated from FHHand Nr4a12/2 rats at weeks 4, 8, 12, and 20. At weeks 4–8,there was no difference in immune response (i.e., number ofcells isolated from intraperitoneal cavity). However, at weeks 12–20, there was a more significant cellular response in Nr4a12/2

rats compared with FHH controls (Figure 4A). Gene expressionchanges in important genes that characterizeM1orM2polarizedmacrophages were studied at each time point (SupplementalFigure 8A). Macrophages isolated from Nr4a12/2 rats demon-strated downregulation of Il10, Il12b, and Tnf-a and upregula-tion of Nos2 fromweeks 4 to 8, but by week 12 or 20 only Nos2was significantly changed (increased inNr4a12/2 rats). Kruppel-like factor 4 was significantly downregulated and suppressor ofcytokine signaling 3 (Socs3) was upregulated.

Macrophages isolated at week 20 were selected for additionalstudy because this time frame (weeks 16–24) demonstrated themost significant renal injury based onproteinuria, histology, anddecline in kidney function. Macrophages isolated from bothgroups were cultured with or without LPS. Whole genome mi-croarray analysis of LPS-treated macrophages from Nr4a12/2

rats demonstrated amore significant upregulation of proinflam-matory immune response genes, including Il-6, Il-12b, Il23,Tnfa, and Nos2 that typical characterize M1 polarized macro-phages, and to a lesser extent several anti-inflammatory genes(Il-10 and Arg1) compared with controls (Figure 4, B and C).Quantitative real-time PCR and evaluation of secreted cytokinesand chemokines (measured by ELISA) confirmed the microa-rrayfindings that LPS-treatedmacrophages isolated fromNr4a12/2

rats exhibited more robust proinflammatory immune activa-tion compared with those isolated from FHH controls (Figure4, C and D). Similar to the thioglycolate-elicited macrophages,Nr4a12 /2 macrophages derived from bone marrowdemonstrated a significant upregulation of Il-6, Il-12b, Tnfa,and Nos2 compared with FHHmacrophages upon stimulationwith LPS (Supplemental Figure 8B).

Table 1. Summary of body weight, organ weights, blood, and kidney function parameters in FHH and Nr4a12/2 rats at weeks8, 16, and 24

TraitWeek 8 Week 16 Week 24

FHH Rats Nr4a12/2 Rats FHH Rats Nr4a12/2 Rats FHH Rats Nr4a12/2 Rats

Body weight (g) 20763.1 19062.6a 35265.8 32865.2a 42969.5 352614.0a

Kidney weight (mg/g) 9.260.27 10.360.14a 9.260.27 10.360.14a 8.160.18 11.060.53a

Heart weight (mg/g) 3.760.23 3.960.8 3.760.23 3.960.8 3.160.06 3.460.13Liver weight (mg/g) 35.660.73 42.761.56a

Drinking volume (ml) 4864.0 3863.4 3666.2 2864.4 2065.0 2262.4Urine volume (ml) 4763.4 3762.1 3965.3 3163.7 2463.7 2462.1Albuminuria (mg/24 h per 100 g body wt) 0.660.09 862.3.5a 1266.1 3265.5a 1763.6 5265.8a

Creatinine clearance (ml/minper g kidney weight)

1.260.07 1.060.12 0.960.06 0.760.06 1.260.2 0.660.2a

BUN (mg/dl) 1461.5 1460.6 1460.6 1760.6a 1461.6 2361.6a

Cholesterol 7064.7 7162.1 8467.9 12268.7a 10269.7 283629.4a

Triglycerides 3261.4 3063.0 3765.3 82612.6a 5064.4 192643.3a

aP,0.05.

2502 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 2499–2510, 2014

BASIC RESEARCH www.jasn.org

DISCUSSION

In this study, we aimed to determine the role of Nr4a1 in theprogression of kidney injury using the FHH rat, which isknown to develop hypertension, proteinuria, and glomerular

disease.11–13 Time course evaluation of re-nal injury and renal function in FHH ratsdeficient in Nr4a1 (Nr4a12/2) demon-strated that loss of Nr4a1 leads to early on-set of kidney injury and progressive declinein kidney function compared with wild-type FHH rats and this effect is independentof changes in systemic or renal hemodynam-ics. Histologically, kidneys from FHH andNr4a12/2 rats exhibited a similar degree ofglomerular and vascular injury that be-came more severe over the course of study.By contrast, tubulointerstitial injury was sig-nificantly increased in Nr4a12/2 rats andwas characterized by macrophageinfiltration and upregulation of inflamma-tory pathways, suggesting a role of im-mune cell infiltration in the pathogenesisof kidney disease in the Nr4a12/2 model.In support of these findings, transplanta-tion of bone marrow from wild-type FHHinto Nr4a12/2 rats resulted in an almostcomplete rescue of proteinuria and renalfunction phenotypes by week 24. In vitrostudies using macrophages isolated fromNr4a12/2 rats demonstrated significant up-regulation of proinflammatory immune re-sponse genes and secretion of chemokines/cytokines upon stimulation with LPS com-pared with controls. In total, these datasuggest that the loss of Nr4a1 in cells ofhematopoietic origin (e.g., lymphocytes)is the primary mechanism of the kidneyinjury in the Nr4a12/2 rats.

The enhanced kidney injury observed inNr4a12/2 rats is likely caused, in part, bythe FHH genetic background, which is per-missive for the development of kidney in-jury. The mechanism of kidney injury inthe FHH rats (summarized in Figure 5) isproposed to occur primarily through lackof RBF autoregulation that results in trans-mission of elevated systemic BP to the glo-merulus, promoting increased capillarypressure and glomerular injury.14,15 Theglomerular injury results in an increase infiltered protein that in combination withtubular defects impairs reuptake of filteredprotein16 and leads to additional tubularinjury and recruitment of inflammatory

cells (monocytes). Thus, the FHH genetic background pro-vides an environment inwhich loss of Nr4a1 enhances inflam-matory processes that are already underway, leading to moreinjury in the kidney and culminating in an additional loss ofrenal function (Figure 5).

Figure 2. Temporal evaluation of renal injury in FHH and Nr4a12/2 rats at weeks 8, 16, and24. (A) Glomerular, tubulointerstitial, and vascular injury. Each glomerulus is scored ona scale from 0 (normal) to 4 (global sclerosis). A minimum of 20 glomeruli are scored foreach kidney section (n=6 per group/time). For tubulointerstitial injury (fibrosis), a minimumof 20 random images per kidney section are studied (n=6 animals per group). Vascularinjury is assessed by measurement of vessel wall thickening. Vessel wall thickening (vesselmedia, micrometers squared) is calculated by measuring the outer circumference of thevessel minus the inner circumference of the lumen (20 random images per animal, n=6animals per group). (B) Representative images of kidney sections from FHH and Nr4a12/2

rats at week 24. (C) Quantification of macrophage (ED-1–positive cells) infiltration in glo-meruli (weeks 8, 16, and 24) and representative immunohistochemistry images at week 24.(D) Macrophage infiltration in tubulointerstitial region (weeks 8, 16, and 24) and repre-sentative immunohistochemistry images at week 24. *P,0.05 compared with FHH.

J Am Soc Nephrol 25: 2499–2510, 2014 Loss of Nr4a1 and Kidney Injury 2503

www.jasn.org BASIC RESEARCH

Kidney injury is a complex pathophysio-logic process that involves a number ofdifferent types of immune cells and pro-cesses. In particular, monocytes/macro-phages play a pivotal role in regulating thefunction of the immune system and theirdysfunction can lead to chronic inflamma-tion.17 Macrophages are typically classifiedinto two distinct types: M1, whichrepresent a proinflammatory state, and M2(with additional subcategoriesM2a, b, and c),which exhibit anti-inflammatory func-tions. A disruption in the balance betweenthe two can lead to the sustained activationof M1 and predispose to chronic inflamma-tory states.17 Our data demonstrate that lossof Nr4a1 leads to a robust macrophage re-sponse in kidneys from Nr4a12/2 rats,which correspondswith progressive protein-uria and renal function decline. Although,we did not specifically assess the degree ofM1 or M2 macrophages in this study, thegene expression profiles of unstimulatedisolated macrophages at multiple timepoints suggest differential regulation ofIl10, Il12b, Tnf-a, and Nos2 betweenFHH- and Nr4a12 /2 -derived macro-phages. In addition, downregulation ofKruppel-like factor 4 and upregulation ofSocs3, both observed in Nr4a12/2 unstimu-lated macrophages, were previously associ-ated with M1 macrophage activation.18,19

LPS-stimulated macrophages fromNr4a12 /2 rats (either thioglycolate orbone marrow derived) demonstrate a sig-nificant and robust M1 macrophage re-sponse characterized by increased Il6,Il12b, Tnf-a, and Nos2 expression andprotein levels compared with FHH rats.Contrary to these findings, LPS-stimulatedmacrophages also resulted in increased Il10expression, an anti-inflammatory cyto-kine. This was unexpected, but may be aconsequence of a mixed population thatincludes M2 polarized macrophages. In to-tal, in vivo studies demonstrate significantmacrophage infiltration that correlateswith kidney injury and in vitro studiesdemonstrate that loss of Nr4a1 may resultin an imbalance in M1 and M2 macro-phages, with M1 macrophages demon-strating enhanced immune responseswhen stimulated.

NR4A receptors were first studied in thebrain and early studies suggested a role in

Figure 3. Bone marrow crosstransplantation studies in FHH and Nr4a12/2 rats. (A andB) Proteinuria and renal function in FHH or Nr4a12/2 rats undergoing reciprocal bonemarrow transplantation. Recipient animals are irradiated (irr) at week 8, provided bonemarrow (bm) isolated from donor animals, and evaluated for proteinuria and/or renalfunction markers at weeks 12, 16, 20, and 24. (C) Morphometric analysis of tubu-lointerstitial injury (fibrosis) and representative Masson’s trichrome images. (D)Quantification of tubulointerstitial macrophage infiltration and immunohistochemistrykidney images from FHH and Nr4a12/2 bone marrow transplant animals at week 24.*P,0.05 versus control groups; †P,0.05 versus other groups. BW, body weight; KW,kidney weight.

2504 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 2499–2510, 2014

BASIC RESEARCH www.jasn.org

Figure 4. Thioglycolate-elicited macrophages and primary cell culture from FHH and Nr4a12/2 rats. (A) Quantitation of intraperitonealcell infiltration at weeks 4, 8, 12, and 20. (B) Hierarchical clustering and gene ontology of differentially expressed genes between LPS-treated cultured macrophages (Mɸ) from FHH and Nr4a12/2 rats at week 20. Mɸ FHH-LPS is set as the control. The heat map colorsdenoted how genes are downregulated or upregulated within each group and across groups (FHH versus Nr4a12/2). Genes denoted in

J Am Soc Nephrol 25: 2499–2510, 2014 Loss of Nr4a1 and Kidney Injury 2505

www.jasn.org BASIC RESEARCH

apoptosis and proliferation.20 Subsequently, in vitro and invivo studies in metabolic tissues, including skeletal muscle,adipose, and liver cells, identified an association between theNR4A subgroup and specific aspects of lipid, carbohydrate,and energy homeostasis.21 For example, Nr4a1 (Nur77)2/2

mice were shown to exhibit insulin resistance (muscle andliver), altered glucose metabolism, and increased lipid levelsthat resulted in hepatic steatosis.22 Increased lipid levels andhepatic steatosis were also observed in this study, suggestingthat the observed metabolic changes in the Nr4a12/2 modelare likely independent from the observed decline in kidneyfunction.

The role of Nr4a1 (Nur77) in vascular disease, namelyatherosclerosis, has been extensively investigated.4,9 LikeCKD,atherosclerosis involves a significant inflammatory compo-nent that is characterized by infiltration of monocytes (in re-sponse to endothelial activation) as well as differentiation intomacrophages that release cytokines/growth factors that aggra-vate inflammation and lead to activation of smooth musclecells that proliferate into the lesion.23 A recent study foundthat mice transplanted with Nr4a1-deficient bone marrowexhibited a significant increase in atherosclerosis comparedwith wild-type bone marrow transplanted controls.24 Theseanimal studies as well as cell culture experiments (exposure toLPS) demonstrated that macrophages deficient in Nr4a1 leadsto a proinflammatory phenotype (polarization toward M1) andexacerbates the development of atherosclerotic lesions.25 Inaddition, in vitro studies that performed knockdown of Nr4a1found enhanced macrophage activation demonstrated by in-creased inflammatory cytokine expression.26 Macrophagesstimulated with LPS or INF-g assume an M1 proinflamma-tory phenotype characterized by a high expression level ofNos2, a high capacity to present antigen, and production ofproinflammatory cytokines such as TNF-a and IL-1b. Con-versely, overexpression of Nr4a1 in human THP-1 and U937macrophages was observed to lead to decreased inflammatorycytokines after stimulation with LPS (IL-1b, IL-6, IL-8, macro-phage inflammatory protein-1a, macrophage inflammatoryprotein-1b, and monocyte chemoattractant protein-1).26 Ourbone marrow crosstransplantation studies, in vitromacrophageassessment, and proposed mechanism of kidney injury (Figure5) are consistent with the conclusions reached by atherosclerosisexperiments and in vitro macrophage studies (i.e., that loss ofNr4a1 inmacrophages likely plays a prominent role in observedpathology).

To our knowledge, this is the first study to investigate thepotential role of Nr4a1 in the context of CKD. There is a singlereport that describes the loss of Nr4a1 (in mice) in promoting

epithelial apoptosis and inflammation after ischemia-reperfusioninjury.27 In this case, loss of Nr4a1 in the kidney itself appearsto play a significant role in the observed injury and althoughwe cannot eliminate a possible role of loss of Nr4a1 in thekidney itself, an immune-mediated mechanism appears to bethe predominant mechanism in our CKDmodel. This apparentpleiotropic role of Nr4a1 is consistent with functional studiesdemonstrating both a pro- and anti-inflammatory role forNr4a1 that is dependent on acute versus chronic expressionand cell type.21 Previouswork on the genetic basis ofGN identifiedJund, another transcription factor, with macrophage activationand GN susceptibility in the WKY rat.28 This study providesadditional evidence that transcription factors may play a vitalrole in macrophage activation status and may promotechronic inflammatory responses.

In summary, our data suggest that loss of Nr4a1 in cells ofhematopoietic origin (e.g., macrophages) promotes increasedkidney injury and reduced renal function observed in theNr4a12/2 model. However, the ability of Nr4a1-deficientmacrophages to exacerbate injury in the kidney is likely de-pendent on the predisposition of the genetic background(FHH) to exhibit established renal injury and inflammation.This work establishes that Nr4a1, through immune activation,can have a significant role in promoting kidney injury andfuture studies aimed at enhancing Nr4a1 expression or func-tion could provide a novel therapeutic strategy to treat kidneydisease.

CONCISE METHODS

Animals and Study DesignFHH and Nr4a12/2 rats were obtained from the Medical College of

Wisconsin. Nr4a12/2 rats were originally generated using an ENU

mutagenesis strategy (N-ethyl-N-nitrosourea) on the genetic back-

ground of the FHH rat through the PhysGen Program (http://pga.

mcw.edu) at the Medical College of Wisconsin. All experiments were

approved by the institutional animal care and use committees at the

Medical College of Wisconsin and the University of Mississippi Med-

ical Center.

Protocol 1: Association of Genetic Mutation with ProteinuriaAn F2 segregating population was developed to validate that the ge-

netic mutation (i.e., knockout) was associated with renal injury (pro-

teinuria).29 Male FHH animals were crossed to Nr4a12/2 female rats

to produce F1(FHH3Nr4a12/2) animals. F1 animals were inter-

crossed to produce the F2(FHH3Nr4a12/2) (n=175). Proteinuria

was measured at weeks 8 and 12 after 24 hours urine collection in

blue are downregulated andgenesdenoted in red are upregulated. (C)Quantitative real-timePCR confirmation of important inflammatorygenes identified by microarray on untreated and LPS-treated macrophages from each group. (D) Measurement of secreted inflammatorychemokines/cytokines by ELISA. n=6 for animal macrophage isolation. n=3–4 independent samples for primary culture studies. *P,0.05versus FHH. RANTES, regulated upon activation, normal T cell expressed and secreted.

2506 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 2499–2510, 2014

BASIC RESEARCH www.jasn.org

metabolism cages as previously described.30 Nr4a12/2 were signifi-

cantly smaller in size compared with FHH controls and a significant

correlation was observed between proteinuria and body weight.

Thus, it was necessary to normalize proteinuria for differences in

body weight and it is reported as mg/24 hours per 100 g body wt.

Protocol 2: Temporal Changes in Renal and CardiovascularTraitsAge-matched male Nr4a12/2 rats (n=28) and FHH rats (n=28) were

studied for telemetry-measured BP (n=8), proteinuria, renal func-

tion, and renal injury at 4, 8, 12, 16, and 20weeks-of-age. At each time

point, six animals per group were euthanized to measure body, heart,

kidney, and liver weights and the remainder of animals were aged to

the next time point. Kidney, heart, and liver samples were processed

for histologic examination and serum samples were obtained from

cardiac puncture to measure blood parameters as previously de-

scribed.30,31 A separate group of Nr4a12/2 rats (n=11) and FHH

rats (n=11) were grown to week 24 for additional measurements.

Protocol 3: BP and Renal HemodynamicsAge-matchedmale Nr4a12/2 and FHH rats were raised until week 16

(n=4–6 per group) and week 24 (n=6 per group). Mean arterial

pressure, RBF, autoregulation, and GFR were

measured as previously described.30

Protocol 4: Bone Marrow TransplantationStudiesBonemarrow transplant studieswere performed

on FHH and Nr4a12/2 animals. Donor animals

were euthanized at week 8 with an overdose of

pentobarbital. Femurs were removed from do-

nor rats, either FHH (n=6) and Nr4a12/2

(n=6). Bone marrow was flushed from the fe-

mur using RPMI cell culture media containing

10% FBS (Sigma-Aldrich), 100 U/ml penicillin,

and 100 g/ml streptomycin (Invitrogen). Cells

were centrifuged under low-speed (2503g),

washed, and resuspended in fresh medium at

53107 cells/ml. Recipient animals were irradi-

ated with approximately 6 Gy at 2.56 Gy/min

(approximately 2.00 minutes) using g rays

from a cesium source irradiator (JL Shephard).

Recipient animals were placed under anesthesia

using 2%–3% isoflurane and were administered

an intravenous injectionof 0.2ml of bonemarrow

cell preparation by tail vein within 2 hours of ir-

radiation. Recipient animals were divided into the

following four groups: (1) FHHbm to FHHirr

(control; n=8), (2) FHHbm to Nr4a1irr2/2

(n=7), (3) Nr4a1bm2/2 to Nr4a1irr

2/2 (control;

n=8), and (4) Nr4a12/2bm to FHHirr (n=8). Rats

were allowed to recover for 4weeks to reconstitute

their bone marrow, at which time the animals

were evaluated (week 12) for measures of renal

injury (proteinuria) and again at weeks 16, 20,

and 24. DNA isolated from the spleen was genotyped for presence of

the wild-type or mutation in exon 1 (Nr4a1) to confirm that bone

marrow was successfully transplanted. FHHbm to Nr4a1irr2/2 animals

were found to be heterozygous (i.e., FHH and Nr4a1 genotype).

However, we were unable to confirm the successful transplant of

Nr4a1bm2/2 to FHHirr, suggesting that the loss of Nr4a1 may have

an effect on the ability of the Nr4a1-derived bone marrow cells to es-

tablish themselves in the context of wild-type hematopoietic cells. Thus,

the Nr4a1bm2/2 to FHHirr group was not included in the study.

Protocol 5: Macrophage Isolation and Cell CultureMale FHH and Nr4a12/2 rats (n=6 per group/each time) were given

an intraperitoneal injection of 4% thioglycolate (Sigma-Aldrich) so-

lution in PBS (Thermo Fisher Scientific) at weeks 4, 8, 12, and 20 as

previously described by others.32 At day 4 postinjection, animals were

euthanized under 2%–3% isoflurane gas. PBS containing 3% FBS

(Invitrogen) was injected into the peritoneal cavity. The abdomen

was massaged briefly to release any adherent cells and a small incision

was created in the lower portion of the abdomen and the fluid was

removed using a sterile transfer pipet. Cells were collected by centri-

fugation, resuspended in sterile media, and counted using a cytom-

eter (USA Scientific). For cell culture, approximately 4–63106 cells

Figure 5. Proposed pathophysiologic mechanism of enhanced immune kidney injuryin Nr4a12/2 compared with FHH rats. Wild-type FHH animals exhibit a genetic pre-disposition to renal injury via the following: (1) lack of RBF autoregulation, (2) in-creased capillary pressure, (3) glomerular injury, (4) protein overload and tubulardefects, and (5) recruitment of inflammatory cells. This mechanism, including lack ofautoregulation, tubular defects, and inflammation, promotes the progressive injuryand reduced renal function (6 and 7) observed in the FHH. Loss of Nr4a1 on the FHHgenetic background provides an environment in which enhanced immune activation(shift toward M1 macrophages and possibly other immune cells) exacerbates and/oraccelerates inflammatory processes that lead to more injury, culminating in an additionalreduction in renal function compared with FHH. *CCL2 also known or monocyte chemo-attractant protein-1 (MCP-1) was the top differentially expressed gene and upregulated inkidney from Nr4a12/2 compared with FHH rats (Supplemental Figure 6). WT, wild type;NO, nitric oxide.

J Am Soc Nephrol 25: 2499–2510, 2014 Loss of Nr4a1 and Kidney Injury 2507

www.jasn.org BASIC RESEARCH

(week 20) were placed in sterile, nontissue culture 100 mm315 mm

petri dishes with DMEM/F12 media (Invitrogen) supplemented with

10% FBS. Cells were incubated at 5% CO2/37°C in an incubator for 2

hours to allow macrophages to adhere. Nonadherent B and T cells

were washed away using sterile media after the incubation. Cells were

either (1) treated with 1 mg/ml LPS (Escherichia coli 055:B5; Sigma-

Aldrich) in DMEM/F12/10% FBS solution for 18 hours or (2) un-

treated in DMEM/F12/10% FBS solution for 18 hours (control).

After incubation, the supernatant was removed and stored in 96-well

plates at 280°C for later analysis of cytokine/chemokine production

using an ELISA (Qiagen). Bone marrow–derived macrophages were

derived from FHH and Nr4a12/2 rats as previously described by

others.28 Bone marrow was isolated from animals similar to protocol 4,

and total bonemarrow cells were counted, plated, and cultured for 5 days

using DMEM (Gibco), heat inactivated FBS (Invitrogen), macrophage

colony-stimulating factor, (ProSpec Bio), and penicillin/streptomycin

(Invitrogen) in a 37°C/5% CO2 environment. Adherent cells were then

treated with DMEM/F12-10 containing LPS (E. coli 055:B5; Sigma-

Aldrich) or DMEM/F12-10 media alone for 4 hours. Macrophages

were removed using sterile PBS and a cell scraper and were processed

for RNA. RNA was used for gene expression using the Affymetrix

GeneAtlas system or real-time PCR.

Histology and ImmunohistochemistryKidneys were fixed in 10%buffered formalin, embedded in paraffin, cut

into 4-mmsections and stainedwith hematoxylin and eosin andMasson’s

trichrome (n=5–6 per group under each protocol/time point). Samples

processed for electron microscopy were processed as previously

described.33 Kidney sections were evaluated by light microscopy for the

presence of glomerular injury (n=20 glomeruli per kidney section) in-

cluding the degree of glomerulosclerosis and mesangial expansion as

previously described.31,33 Interstitial fibrosis was determined by evalu-

ation of slides stained with Masson’s trichrome to quantify the percent

fibrosis (blue staining) compared with the background. Tubular injury

was assessed by degree of tubular atrophy, vacuolization, dilation, and/

or protein casts on a scale from 0 (normal) to 4 (global sclerosis) as

previously described.34 Vascular injury was assessed by wall thickening

and cellular proliferation.Vesselwall thickening (vesselmedia, inmicro-

meters squared)was calculated bymeasuring the outer circumference of

the vessel minus the inner circumference of the lumen (20 random

images at340 per rat).

Macrophage, T-cell, and B-cell infiltration was assessed by immu-

nohistochemistry on unstained sections using primary antibodies

directed at CD68/ED-1, CD22, and CD43 (Santa Cruz Biotechnology)

and detected by 3,39-diaminobenzidine (Ultravision LPValue Detection

System; Thermo Fisher Scientific).30 Slides were counterstained with

methyl green (n=4 sections per group; 15–20 images). Images were

captured using a Nikon 55i microscope with a DS-Fi1 5-Meg Color C

digital camera (Nikon, Melville, NY) and were analyzed using Nis-

Elements image analysis software (version 3.03; Nikon).

Molecular MethodsGenotyping and SequencingGenomicDNAwas obtained by tail biopsy and prepared usingWizard

SV 96 Genomic DNA kit (Promega, San Luis Obispo, CA). Genotyping

was done using standard PCR-ethidium bromide-gel electrophoresis.

The point mutation in exon 1 (Nr4a1) created an AseI restriction site in

theNr4a12/2 thatwasused for genotyping segregating population (Sup-

plemental Figure 1). Primers were designed to amplify across the point

mutation in exon 1 (Nr4a1-F, 59-TCGTCCTCGGCCACCTCTCC-39;

Nr4a1-R, 59-GCCTTCATAAGTCTGGCTGGGGG-39). DNA sequenc-

ing was performed on a CEQ8000 using the DTCS Quick Start Kit

(BeckmanCoulter, Brea,CA). Sequencing readswere assessed for quality

andaligned to theBNreference sequence using theDNASTARLasergene

v7.2 software package.

Microarray and Real-Time PCRWhole genome transcript analysis was performed using Affymetrix

platforms (kidney tissue collected under protocol 1) from FHH and

Nr4a12/2 rats at weeks 8 (n=3 per group), 16 (n=3), and 24(n=3).

RNA was isolated using TRIzol and Invitrogen PureLink Kit (Life

Technologies) and evaluated for quality and integrity (Bio-Rad Ex-

perion System) as previously described.35 Kidney RNA time course

samples were processed per the manufacturer’s directions for specific

application (GeneChip 1.0 ST) using Affymetrix equipment (Scanner

3000 7G System) or primary macrophage cultures (GeneAtlas). Hy-

bridized chips were automatically washed, stained, and scanned at the

University of Mississippi Medical Center Institutional Molecular and

Genomics Core using Affymetrix equipment. Data obtained from

these gene expression studies are deposited in the Gene Expression

Omnibus database (accession number GSE54714; http://www.ncbi.

nlm.nih.gov/geo).

Analysis of microarray data was performed using software

provided by Affymetrix (Affymetrix Expression Console Software)

and the commercially available GeneSifter software platform

(http://www.genesifter.net) and/or Array Star 5 (DNASTAR Inc.,

Madison,WI). In brief, differentially expressed genes evaluated by a

t test using two methods: (1) family-wise error rate procedure,

using P,0.05 and a fold change $61.2; and/or (2) Benjamani

and Hochberg false discovery rate, which corrects for multiple

comparison, using P,0.05 and a fold change $61.2. Two-way

ANOVAusing a P,0.01 or lower was performed to identify strain3

time interaction effects between groups. Gene networks and func-

tional analysis were evaluated using Ingenuity Pathways Analysis

(http://www.ingenuity.com; Ingenuity Systems). Gene expression

differences were confirmed using SYBR Green dye chemistry on

Bio-Rad CFX96 (n=6–8 per group). RNA was reverse-transcribed

to cDNA using an iScript cDNA Synthesis Kit and real-time PCR

(Bio-Rad) was performed using SsoFast EvaGreen Supermix (Bio-Rad).

Predesigned PrimeTime quantitative PCR primers were purchased

from IDT to assess gene expression levels of chemokines, cytokines,

and growth factors.

Statistical AnalysesLinkage study associations between genotype and phenotype were

assessed for the Nr4a1 locus using ANOVAwithin the SPSS program

(Chicago, IL). An independent t test or one-ANOVA followed by

Bonferroni correction for multiple comparisons was used for de-

scribed animal experiments. A P,0.05 was considered statistically

significant. All data are presented as the mean6SEM.

2508 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 2499–2510, 2014

BASIC RESEARCH www.jasn.org

ACKNOWLEDGMENTS

M.R.G is supported by the National Institutes of Health National

Heart, Lung, and Blood Institute (HL094446), the Robert M. Hearin

Foundation, and the Research Center of Excellence in Pediatric Ne-

phrology (RCEPN) at the Medical College of Wisconsin. K.R.R is

supported by funds from RCEPN and the Clinical and Translational

Science Institute at the Medical College of Wisconsin. The work per-

formed through theUniversity ofMississippiMedical CenterMolecular

andGenomics Facility is supported, in part, by funds from the National

Institutes of Health National Institute of General Medical Sciences,

including the Mississippi IDeA Networks of Biomedical Research Ex-

cellence (P20GM103476), Center for Psychiatric Neuroscience–Center

of Biomedical Research Excellence (P30GM103328) and Obesity,

Cardiorenal, andMetabolic Diseases–Center of Biomedical Research

Excellence (P20GM104357). The content of this article is solely the

responsibility of the authors and does not necessarily represent the

official views of the National Institutes of Health.

DISCLOSURESNone.

REFERENCES

1. US Renal Data Service: USRDS 2011 Annual Data Report: Atlas ofChronic Kidney Disease and End-Stage Renal Disease in the UnitedStates. Bethesda, MD, National Institutes of Health, National Instituteof Diabetes and Digestive and Kidney Diseases, 2011

2. Coresh J, Selvin E, Stevens LA, Manzi J, Kusek JW, Eggers P, Van LenteF, Levey AS: Prevalence of chronic kidney disease in the United States.JAMA 298: 2038–2047, 2007

3. Trivedi H: Cost implications of caring for chronic kidney disease: Are in-terventions cost-effective? Adv Chronic Kidney Dis 17: 265–270, 2010

4. Pearen MA, Muscat GEO: Minireview: Nuclear hormone receptor 4Asignaling: Implications formetabolic disease.Mol Endocrinol 24: 1891–1903, 2010

5. Martínez-González J, Badimon L: The NR4A subfamily of nuclear re-ceptors: New early genes regulated by growth factors in vascular cells.Cardiovasc Res 65: 609–618, 2005

6. Mohan HM, Aherne CM, Rogers AC, Baird AW,Winter DC, Murphy EP:Molecular pathways: The role of NR4A orphan nuclear receptors incancer. Clin Cancer Res 18: 3223–3228, 2012

7. Kanzleiter T, Wilks D, Preston E, Ye J, Frangioudakis G, Cooney GJ:Regulation of the nuclear hormone receptor nur77 in muscle: Influenceof exercise-activated pathways in vitro and obesity in vivo. BiochimBiophys Acta 1792: 777–782, 2009

8. Fu Y, Luo L, Luo N, Zhu X, Garvey WT: NR4A orphan nuclear receptorsmodulate insulin action and the glucose transport system: Potential rolein insulin resistance. J Biol Chem 282: 31525–31533, 2007

9. Zhao Y, Bruemmer D: NR4A orphan nuclear receptors: Transcriptionalregulators of gene expression in metabolism and vascular biology.Arterioscler Thromb Vasc Biol 30: 1535–1541, 2010

10. McMorrow JP, Murphy EP: Inflammation: A role for NR4A orphan nu-clear receptors? Biochem Soc Trans 39: 688–693, 2011

11. Kuijpers MH, Gruys E: Spontaneous hypertension and hypertensiverenal disease in the fawn-hooded rat. Br J Exp Pathol 65: 181–190,1984

12. Simons JL, Provoost AP, Anderson S, Troy JL, Rennke HG, SandstromDJ, Brenner BM: Pathogenesis of glomerular injury in the fawn-hoodedrat: Early glomerular capillary hypertension predicts glomerular scle-rosis. J Am Soc Nephrol 3: 1775–1782, 1993

13. MattsonDL, Dwinell MR,GreeneAS, Kwitek AE, Roman RJ, CowleyAWJr, Jacob HJ: Chromosomal mapping of the genetic basis of hyper-tension and renal disease in FHH rats. Am J Physiol Renal Physiol 293:F1905–F1914, 2007

14. VanDokkumRPE, Alonso-GaliciaM, Provoost AP, JacobHJ, Roman RJ:Impaired autoregulation of renal blood flow in the fawn-hooded rat.Am J Physiol 276: R189–R196, 1999

15. van Dokkum RPE, Sun C-W, Provoost AP, Jacob HJ, Roman RJ: Alteredrenal hemodynamics and impaired myogenic responses in the fawn-hooded rat. Am J Physiol 276: R855–R863, 1999

16. Rangel-Filho A, Lazar J, Moreno C, Geurts A, Jacob HJ: Rab38 modu-lates proteinuria in model of hypertension-associated renal disease. JAm Soc Nephrol 24: 283–292, 2013

17. Murray PJ, Wynn TA: Protective and pathogenic functions of macro-phage subsets. Nat Rev Immunol 11: 723–737, 2011

18. Liao X, Sharma N, Kapadia F, Zhou G, Lu Y, Hong H, Paruchuri K,Mahabeleshwar GH, Dalmas E, Venteclef N, Flask CA, Kim J, DoreianBW, Lu KQ, Kaestner KH, Hamik A, Clément K, Jain MK: Krüppel-likefactor 4 regulates macrophage polarization. J Clin Invest 121: 2736–2749, 2011

19. Arnold CE, Whyte CS, Gordon P, Barker RN, Rees AJ, Wilson HM: Acritical role for SOCS3 in promoting M1 macrophage activation andfunction in vitro and in vivo. Immunology 141: 96–110, 2014

20. Watson MA, Milbrandt J: The NGFI-B gene, a transcriptionally in-ducible member of the steroid receptor gene superfamily: Genomicstructure and expression in rat brain after seizure induction. Mol CellBiol 9: 4213–4219, 1989

21. Maxwell MA, Muscat GE: The NR4A subgroup: Immediate early re-sponse genes with pleiotropic physiological roles. Nucl Recept Signal4: e002, 2006

22. Chao LC, Wroblewski K, Zhang Z, Pei L, Vergnes L, Ilkayeva OR, DingSY, Reue K, Watt MJ, Newgard CB, Pilch PF, Hevener AL, Tontonoz P:Insulin resistance and altered systemic glucose metabolism in micelacking Nur77. Diabetes 58: 2788–2796, 2009

23. Woollard KJ, Geissmann F: Monocytes in atherosclerosis: Subsets andfunctions. Nat Rev Cardiol 7: 77–86, 2010

24. Hamers AAJ, Vos M, Rassam F, Marinkovi�c G, Kurakula K, van Gorp PJ,de Winther MP, Gijbels MJ, de Waard V, de Vries CJ: Bone marrow-specific deficiency of nuclear receptor Nur77 enhances atherosclerosis.Circ Res 110: 428–438, 2012

25. Hanna RN, Shaked I, Hubbeling HG, Punt JA,WuR, Herrley E, ZauggC,Pei H, Geissmann F, Ley K, Hedrick CC: NR4A1 (Nur77) deletion po-larizes macrophages toward an inflammatory phenotype and increasesatherosclerosis. Circ Res 110: 416–427, 2012

26. Bonta PI, van Tiel CM, Vos M, Pols TW, van Thienen JV, Ferreira V,Arkenbout EK, Seppen J, Spek CA, van der Poll T, Pannekoek H, deVries CJ: Nuclear receptors Nur77, Nurr1, and NOR-1 expressed inatherosclerotic lesion macrophages reduce lipid loading and in-flammatory responses. Arterioscler Thromb Vasc Biol 26: 2288–2294,2006

27. Balasubramanian S, Jansen M, Valerius MT, Humphreys BD, StromTB: Orphan nuclear receptor Nur77 promotes acute kidney injuryand renal epithelial apoptosis. J Am Soc Nephrol 23: 674–686,2012

28. Behmoaras J, Bhangal G, Smith J,McDonald K,Mutch B, Lai PC, DominJ, Game L, SalamaA, Foxwell BM, PuseyCD, CookHT, Aitman TJ: Jundis a determinant of macrophage activation and is associated with glo-merulonephritis susceptibility. Nat Genet 40: 553–559, 2008

29. Garrett MR, Dene H, Rapp JP: Time-course genetic analysis of albu-minuria in Dahl salt-sensitive rats on low-salt diet. J AmSocNephrol 14:1175–1187, 2003

J Am Soc Nephrol 25: 2499–2510, 2014 Loss of Nr4a1 and Kidney Injury 2509

www.jasn.org BASIC RESEARCH

30. Williams JM, Johnson AC, Stelloh C, Dreisbach AW, Franceschini N,Regner KR, Townsend RR, Roman RJ, Garrett MR: Genetic variants inArhgef11 are associated with kidney injury in the Dahl salt-sensitive rat.Hypertension 60: 1157–1168, 2012

31. Regner KR, Harmon AC, Williams JM, Stelloh C, Johnson AC, KylePB, Lerch-Gaggl A, White SM, Garrett MR: Increased suscepti-bility to kidney injury by transfer of genomic segment from SHRonto Dahl S genetic background. Physiol Genomics 44: 629–637,2012

32. Ray A, Dittel BN: Isolation ofmouse peritoneal cavity cells. J Vis Exp 35:1488, 2010

33. Garrett MR, GunningWT, Radecki T, Richard A: Dissection of a geneticlocus influencing renal function in the rat and its concordance with

kidney disease loci on human chromosome 1q21. Physiol Genomics30: 322–334, 2007

34. Packard M, Saad Y, Gunning WT, Gupta S, Shapiro J, Garrett MR: In-vestigating the effect of genetic background on proteinuria and renalinjury using two hypertensive strains. Am J Physiol Renal Physiol 296:F839–F846, 2009

35. MengH, Garrett MR, DeneH, Rapp JP: Localization of a blood pressureQTL to a 2.4-cM interval on rat chromosome 9 using congenic strains.Genomics 81: 210–220, 2003

This article contains supplemental material online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2013070786/-/DCSupplemental.

2510 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 2499–2510, 2014

BASIC RESEARCH www.jasn.org