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TGF-␣ Mediates Genetic Susceptibility to Chronic Kidney Disease

Denise Laouari,* Martine Burtin,* Aure´lie Phelep,* Carla Martino,* Evangeline Pillebout,* Xavier Montagutelli,† Ge´rard Friedlander,* and Fabiola Terzi*

*INSERM U845, Centre de Recherche “Croissance et Signalisation,” Universite´ Paris Descartes, Hoˆpital Necker Enfants Malades, Paris, France; and †Unite´deGe´ne´ tique Fonctionnelle de la Souris, URA CNRS 2578, Institut Pasteur, Paris, France

ABSTRACT The mechanisms of progression of chronic kidney disease (CKD) are poorly understood. Epidemiologic studies suggest a strong genetic component, but the genes that contribute to the onset and progression of CKD are largely unknown. Here, we applied an experimental model of CKD (75% excision of total renal mass) to six different strains of mice and found that only the FVB/N strain developed renal lesions. We performed a genome-scan analysis in mice generated by back-crossing resistant and sensitive strains; we identified a major susceptibility locus (Ckdp1) on chromosome 6, which corresponds to regions on human chromosome 2 and 3 that link with CKD progression. In silico analysis revealed that the locus includes the gene encoding the EGF receptor (EGFR) ligand TGF-␣. TGF-␣ protein levels markedly increased after nephron reduction exclusively in FVB/N mice, and this increase preceded the develop- ment of renal lesions. Furthermore, pharmacologic inhibition of EGFR prevented the development of renal lesions in the sensitive FVB/N strain. These data suggest that variable TGF-␣ expression may explain, in part, the genetic susceptibility to CKD progression. EGFR inhibition may be a therapeutic strategy to counteract the genetic predisposition to CKD.

J Am Soc Nephrol 22: 327–335, 2011. doi: 10.1681/ASN.2010040356

Human chronic kidney diseases (CKD), regardless and this occurs independently of glycemic control of their etiology, are characterized by progressive or hypertension.1,2 However, the propensity to de- destruction of the renal parenchyma and loss of velop ESRD differs among ethnic groups3–7 and it functional nephrons, leading to ESRD. Approxi- shows familial clustering.7–10 Similarly, the rate of mately 13% of adults suffer from CKD in industri- progression of primary hereditary kidney diseases alized countries and the incidence of ESRD in- can vary among members of the same family,11–13 creases by 6% to 8% per year. Therefore, suggesting that genes unrelated to the disease understanding the pathophysiology of CKD is a key challenge for public health. The mechanisms of CKD progression are poorly Received April 7, 2010. Accepted September 25, 2010. understood. Although clinical studies point to the Published online ahead of print. Publication date available at important role of environmental factors in the bio- www.jasn.org. logic processes leading to renal deterioration, epi- D.L. and M.B. contributed equally to this work. demiologic studies have underscored the impor- Correspondence: Dr. Fabiola Terzi, INSERM U845, Universite´ tance of genetic components. Indeed, it has been Paris Descartes, Team: Mechanisms and therapeutic strategies in chronic nephropathies, Hoˆpital Necker Enfants Malades, observed that the evolution of CKD varies consid- Tour Lavoisier, 6e`me e´ tage, 149 Rue de Se`vres, 75015 Paris, erably among individual patients exposed to the France. Phone: ϩ33 144495245; Fax: ϩ33 144490290; E-mail same risk factors. Only a proportion of patients [email protected] with diabetes or hypertension develop renal failure, Copyright © 2011 by the American Society of Nephrology

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(modifiers) might account for the susceptibility to develop (C57BL/6xDBA/2)F1 (hereafter denoted B6D2F1), and (C57BL/ ESRD. 6xSJL)F1 showed only mild glomerular and tubular hypertrophy Although many studies have sought to discover the modi- (Figure 1A). Neither glomerulosclerosis nor tubulointerstitial le- fiers of CKD progression, the gene variants that predispose sions could be detected up to 6 months after Nx except in a narrow individuals to ESRD remain largely unknown. The genetic scarring area originating from the surgical excision of the two complexity of human populations and the difficulty of stan- poles. Development of renal lesions in FVB/N mice resulted in dardizing analyses of environmental factors in complex dis- severe proteinuria (26.8 Ϯ 5.3 mg/d versus 0.9 Ϯ 0.1 mg/d in Nx eases have hampered the identification of these crucial modi- and control mice, respectively; P Ͻ 0.001) and a moderate in- fiers. Efforts to discover novel modifiers must, therefore, crease in mean arterial BP (145 Ϯ 8mmHgversus 116 Ϯ 5 mmHg include experimental models.14 Numerous animal models in Nx and control mice, respectively; P Ͻ 0.05). In resistant have been developed to elucidate the pathophysiology of CKD. B6D2F1 mice, proteinuria could not be detected up to 2 months Among these, the remnant kidney model is a mainstay because after Nx, whereas moderate hypertension developed (139 Ϯ 5 nephron reduction characterizes the evolution of most human mmHg). Disease progression was rapid in FVB/N mice with com- CKD. Consequently, this model recapitulates many features of plete destruction of the remnant kidney and ESRD that led to a human CKD, including hypertension, proteinuria, and glo- 50% survival rate at 3 months. The clear bimodal phenotype merular and tubulointerstitial lesions. Over the past 50 years, among the susceptible and resistant strains provides an experi- this model has led to the discovery of critical pathways and, mental framework to identify modifiers of CKD progression. more importantly, to the design of therapeutic strategies to slow down CKD progression, that is, the widely clinically used Inheritance Pattern of Renal Deterioration renin-angiotensin system inhibitors.15 More recently, studies To determine the inheritance pattern of lesion susceptibility, in different mouse strains have highlighted the importance of we performed reciprocal intercrosses between FVB/N and genetic factors. In fact, it has been shown that whereas nephron B6D2F1 mice and analyzed the kidneys of their progeny (here- reduction induces early and severe pathologic lesions in ROP after denoted G1) 2 months after Nx. Remnant kidneys of G1 mice, other strains, for example, C57BL/6 or C3H, are resistant progeny were severely damaged, similarly to those of the pa- to early renal deterioration.16–19 Similarly, we showed that the rental FVB/N strain, but exclusively in G1 males (Figure 1B). course and extent of renal lesions after 75% surgical excision of In fact, 96% of males developed severe glomerulosclerosis, tu- renal mass vary significantly between two mouse strains: bular dilation, and interstitial fibrosis, whereas 96% of females whereas the FVB/N mice develop renal lesions, the (C57BL/ were resistant to lesion development. The severity and the ex- 6xDBA/2)F1 are resistant to early deterioration.20 tension of glomerular, tubular, and interstitial lesions were un- Here, we combined this experimental model of CKD, ex- distinguishable from those of the parental FVB/N strain (Fig- perimental crosses, and a whole genome scan to identify a lo- ure 1). This phenotype was observed regardless of the direction cus that confers increased susceptibility to lesion development of the crosses, thereby excluding a sex-linked inheritance. in FVB/N mice. Furthermore, we provide evidence that Complementary reciprocal crosses between either C57BL/6 TGF-␣, a gene of the locus that codes for a ligand of epidermal and FVB/N or DBA/2 and FVB/N mice confirmed these results growth factor receptor (EGFR), is critically involved in the (data not shown). On the basis of the G1 phenotype, we hy- genetic predisposition to CKD progression. pothesized that the FVB/N variant of one or more modifier genes may confer susceptibility to develop renal lesions after Nx in a dominant fashion in males and recessively in females, RESULTS although a reduced penetrance could not be formally ex- cluded. To test this hypothesis, we backcrossed G1 females to Genetic Susceptibility To Develop Renal Lesion after either FVB/N or B6D2F1 males and evaluated renal morphol- Nephron Reduction ogy 2 months after Nx. Among 100 G2 progeny, the percentage To understand how genetic background influences the progres- of mice developing severe lesions (Figure 1B) was very close to sion of CKD, we performed 75% reduction of renal mass (Nx) in the expected values in the two crosses (Table 1), a result con- four different mouse strains and two F1 hybrids and examined sistent with a major recessive modifying locus for the lesion remnant kidneys at 2, 4, and 6 months after surgery. Only one susceptibility trait. As in the parental strains, the renal pheno- strain, FVB/N, displayed severe renal lesions 2 months after Nx. type showed a clear bimodal expression with severe glomerular The most striking feature was diffuse tubular lesions consisting and tubular lesions in the sensitive G2 mice versus mild glo- mostly of dilations with microcyst formation obliterated by pro- merular and tubular hypertrophy in the resistant G2 animals. tein casts and focal area of tubular dedifferentiation. They were associated with glomerular lesions characterized by diffuse mes- Linkage Mapping of a Modifier Locus for CKD angial sclerosis with segmental/global hyalinization or scarring of Progression the tuft in 50% to 70% of glomeruli. Moreover, mild fibrosis and To map the locus that modifies the renal lesion phenotype, a multifocal mononuclear cell infiltration were observed in the in- complete genome scan was performed on females and males terstitium (Figure 1A). In contrast, C57BL/6, DBA/2, 129S2/Sv, from G1xFVB/N and G1xB6D2F1 crosses, respectively, using

328 Journal of the American Society of Nephrology J Am Soc Nephrol 22: 327–335, 2011 www.jasn.org BASIC RESEARCH

A

B

Figure 1. Susceptibility to develop renal lesions after nephron reduction is genetically determined in mice. (A) Renal morphology of control and 75% nephrectomized (Nx) mice 2 and 6 months after surgery in FVB/N and C57BL/6, DBA/2, 129S2/Sv, B6D2F1, and B6SJLF1 parental strains, respectively. Because no differences were detected between control mice, only FVB/N and B6D2F1 strains are shown. PAS staining, magnification, ϫ200. (B) Renal morphology of control and Nx mice 2 months after surgery in G1 male and female and G2 lesion-prone progeny. Because no differences were detected between males and females in G2 lesion-prone progeny, only one G2 mouse is shown. PAS staining, magnification, ϫ200.

64 informative markers distributed across the mouse genome TGF-␣ Is Essential for CKD Progression (average spanning ϭ 20 cM). B6 and D2 alleles were scored as We next try to identify the gene(s) within the locus that might non-FVB for linkage analysis. Data from the two crosses were modify CKD progression. Inspection of the Ckdp1 95% confi- combined and genotypic distributions were matched with the dence interval for potential candidates revealed the presence of putative model for modifier inheritance. Significant linkage TGF-␣, a ligand of EGFR (Supplemental Table 1). Our recent was found only for a locus on chromosome 6 (Figure 2A). findings that TGF-␣ is a crucial intermediate in angiotensin Additional polymorphic markers confirmed significant link- II–induced renal lesions21 prompted us to examine its expres- age (lowest P values) to the 17-cM interval between D6Mit209 sion. TGF-␣ mRNA levels significantly increased in remnant and D6Mit328 (76 to 113 Mpb) (Figure 2B). Hence, a major kidneys of FVB/N mice relative to sham-operated controls, but modifier locus accounts for CKD progression after Nx in the were unchanged in B6D2F1 counterparts (Figure 3A). Simi- mouse; we have designated this locus Ckdp1. larly, TGF-␣ protein levels markedly increased 2 months after

Table 1. Predicted renal lesion frequencies according to our hypothetical model (dominant allele in FVB/N males) versus the observed renal lesion frequencies of G2 male and female progeny Cross G1 Female ؋ FVB/N Male G1 Female ؋ B6D2F1 Male (29 ؍ G2 Female (n (25 ؍ G2 Male (n (34 ؍ G2 Female (n (12 ؍ G2 Male (n Predicted lesions 12 (100%) 17 (50%) 13 (50%) 0 (0%) Observed lesions 11 (92%) 20 (59%) 15 (60%) 3 (10%) G2 mice result from G1 females crossed to either FVB/N or B6D2F1 males. n ϭ number of animals.

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A as compared with that in vehicle-treated animals (Figure 4A). Proteinuria was also significantly decreased (Figure 4B), whereas hypertension was unaffected (Figure 4C).

TGF-␣ Gene Is Not Mutated in FVB/N Strain We next sequenced TGF-␣ cDNA derived from the three pa- rental C57BL/6, DBA/2, and FVB/N strains. Compared with C57BL/6 sequence (http://www.ensembl.org), alignment of TGF-␣ coding sequence revealed only a synonymous single- nucleotide polymorphism (SNP) 81A3G in FVB/N and DBA/2 strains and several 3ЈUTR SNPs (538A3G, 1031A3G, 1316T3C, 2583A3G, 3186G3A) and a polyA 3-bp insertion in the FVB/N and DBA/2 strains. However, all these substitutions are silent mutations. We next screened for regulatory transcriptional mutations in the TGF-␣ gene using a RNA PCR strategy to detect potential allelic differences in B expression of the cognate mRNAs and taking advantage of the neutral polyA 3-bp insertion noted above. To compare relative expression levels of the TGF-␣ allele in FVB/N and C57BL/6 mice, RNA PCR products spanning polymorphic nucleotides were amplified from total RNA prepared from remnant kid- neys of G2 mice. TGF-␣ PCR products showed equivalent ex- pression from both FVB/N and C57BL/6 allele (FVB/N- C57BL/6 ratio: 0.992 Ϯ 0.017, n ϭ 8). Collectively, these results argue against a cis-acting mutation in TGF-␣ gene and indicate that another gene within the locus accounts for lesion suscep- tibility in FVB/N mice by modulating TGF-␣ expression.

DISCUSSION Figure 2. A single locus on chromosome 6 fits in the renal lesion phenotype. (A) Segregation distortion between the lesion phe- The genetic factors that control progression to ESRD are notype in G2 progeny (G1 females crossed to either FVB/N or largely unknown. By applying genetic and molecular ap- B6D2F1 males) by a genomewide scan. Bars represent the max- proaches to a CKD experimental model in six strains of mice, imum distortion found on each chromosome measured by the we have identified a locus on mouse chromosome 6 that is Fischer exact test and plotted as Ϫlog(P). (B) Complete genotyp- significantly linked to the evolution of CKD. More impor- ing data for chromosome 6. Positions are in cM and Mbp as given ␣ by the MGI and Ensembl databases. Discordance indicates the tantly, we have discovered that TGF- , a gene of the locus, is percentage of animals for which the observed phenotype did not crucially involved in the susceptibility to CKD progression. In fit with the expected one. fact, pharmacologic inhibition of TGF-␣ receptor prevented lesion development in the sensitive FVB/N strain, consistent Nx, but exclusively in FVB/N mice (Figure 3B). Immunohis- with TGF-␣ overexpression. However, no mutation could be tochemistry confirmed the increase of TGF-␣ expression detected in the TGF-␣ gene. Collectively, these results identify (Figure 3C) and showed that TGF-␣ protein is mainly lo- a new locus linked to CKD progression and strongly suggest cated in ascending loops of Henle and distal tubules. A care- that genetic variants that control TGF-␣ expression are poten- ful time course analysis revealed that the increase of TGF-␣ tial modifiers of CKD. preceded the development of renal lesions 6 weeks after Nx The experimental model of nephron reduction has been (Figure 3D), suggesting that this molecule may be critically widely used to elucidate the mechanisms underlying the pro- involved in the genetic predisposition of FVB/N mice to gression of CKD.15 In this study, we used this model to identify develop renal lesions. candidates that confer susceptibility to CKD progression. In- To test this hypothesis, we treated FVB/N mice with the terestingly, we showed that among six different strains of mice, selective EGFR tyrosine kinase inhibitor, Iressa, for 2 months only one, the FVB/N, develops severe renal lesions, whereas the after Nx. Interestingly, daily administration of Iressa markedly other ones are resistant to early deterioration. This observation protected remnant kidneys from the deterioration process. In is consistent with previous works showing that C57BL/6 mice fact, the severity of glomerulosclerosis, tubular dilation, and are particularly resistant to proteinuria and glomerulosclerosis interstitial fibrosis were markedly reduced in Iressa-treated mice after Nx.16–19 That 129/Sv mice are resistant to renal deterio-

330 Journal of the American Society of Nephrology J Am Soc Nephrol 22: 327–335, 2011 www.jasn.org BASIC RESEARCH

A B D4Rat95 marker region. This marker has shown significant linkage to urinary albu- min excretion in Munich Wistar Frömter rats,25 a model of progressive CKD. More importantly, our Ckdp1 locus perfectly overlaps with previous reported QTLs in humans. In particular, the locus is ortholo- gous to the human chromosome 2p12-p13 C region, where two recent genomewide as- sociation studies have identified common variants (rs10206899) associated with renal function and CKD progression in several thousands of patients.26,27 In addition, the Ckdp1 locus is syntenic to the human chro- D mosome 3p14–12 and 3q21–22 regions, which have been linked to proteinuria in pa- tients with type 1 diabetic nephropathy.28,29 Of note, several studies have suggested evi- dence of linkage to chromosome 3 also in type 2 diabetic nephropathy.30 The concor- dance among the three species strongly sug- gests that common genetic variants may be involved in CKD progression. On the basis of available mouse genome database (http://www.ensembl.org), we es- tablished that 400 annotated genes were Figure 3. TGF-␣ expression increases before the development of renal lesions in encoded within the candidate Ckdp1 inter- FVB/N mice. (A) TGF-␣ mRNA expression in control (C) and 75% nephrectomized (Nx) val. Inspection for potential candidates B6D2F1 and FVB/N mice, evaluated by ribonuclease protection assay 2 months after revealed that the locus includes the gene surgery. (B) Western blot analysis of TGF-␣ expression in control and Nx B6D2F1 and encoding TGF-␣, a ligand of EGFR. In- ␣Ϫ/Ϫ FVB/N mice. Extracts of TGF- kidneys (KO) served as negative controls. terestingly, previous studies highlighted ␣ (C) Immunostaining for TGF- in control and Nx B6D2F1 and FVB/N mice. Because no the key role of EGFR activation in CKD.31 differences were detected between B6D2F1 and FVB/N control mice, only one group Evidence presented here points to TGF-␣ is shown. Magnification, ϫ200. (D) Western blot analysis of TGF-␣ (upper panel) and renal morphology (lower panel) in control and Nx FVB/N mice 4, 6, and 8 weeks after as the key mediator of the genetic predis- surgery. Because no differences were detected among FVB/N control mice at different position to CKD progression. In fact, we time points, only one group is shown. PAS staining, magnification, ϫ200. Data are demonstrated that inhibition of EGFR means Ϯ SEM; n ϭ 6 for each experimental group. Statistical analysis: ANOVA, prevented renal deterioration in the le- followed by Tukey-Kramer test: Nx versus control: *P Ͻ 0.05; FVB/N versus B6D2F1 sion-prone FVB/N strain. Consistently, mice: #P Ͻ 0.05. we observed that TGF-␣ expression in- creased in FVB/N mice and, more impor- ration might be intriguing because two previous studies have tantly, the increase preceded the development of renal le- reported that this strain may develop proteinuria and mild sions. The increase of TGF-␣ was specific because lesions after Nx.18,19 However, in both studies, the percentage expression of EGF, the principal EGFR ligand synthesized in of Nx was more severe and the follow-up longer than that in the same renal cells, was unchanged by either genetic back- our work, suggesting that the difference in the model used may ground or Nx (data not shown). It is worthy to note that account for this discrepancy. Supporting this idea, we previ- transgenic mice overexpressing TGF-␣ develop severe renal ously observed that mild lesions appear also in the resistant lesions,32,33 whereas transgenic mice broadly expressing EGF B6D2F1 strain 8 months after Nx.22 It is worthy to note that the do not.34 Hence, it is tempting to speculate that genes encoding FVB/N genetic background has been reported to be highly per- molecules involved in regulation of TGF-␣ are potential mod- missive to the onset of renal lesions in other experimental ifiers of CKD progression. Supporting this idea, we previously models of CKD.23,24 showed that inactivation of JunD, a partner of the AP1 tran- Our genome-wide scan analysis revealed a major suggestive scription complex, promotes lesion development upon Nx in locus on chromosome 6 that is associated with the develop- resistant strains by stimulating the expression of TGF-␣.35 No- ment of renal lesions after Nx. Interestingly, the interval con- tably, ontology searches for annotated genes within the Ckdp1 taining the locus is orthologous to the rat chromosome 4 locus revealed 23 genes encoding for transcription factors.

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A binding protein and is of unknown function. Although we can- not rule out the possibility that additional genes are involved because the genome of FVB/N strain has not been completely sequenced, this bioinformatics analysis provides an initial se- lection from 400 genes in the initial Ckdp1 interval to a few potential candidates. The functional analysis of the genetic variants will determine if one of them is the modifier of CKD B C progression in FVB/N mice. In this study, G1 males and females exhibited a significant sexual dimorphism in susceptibility to lesion development af- ter Nx. That this dimorphism was displayed in reciprocal crosses excludes a direct contribution of the X or Y chromo- some to these findings. Several experimental and clinical stud- ies have shown that gender affects the incidence, prevalence and progression of CKD.38 In particular, men are more prone to progressive renal diseases.39 It has been suggested that this disparity may stem from sex hormones,40 but the molecular Figure 4. Pharmacologic inhibition of EGFR prevents lesion de- pathways are not clearly identified. Although elucidating these velopment in FVB/N mice. (A) Renal morphology in control pathways was beyond the scope of this work, preliminary re- (C) and 75% nephrectomized (Nx) FVB/N mice treated with either sults suggest that the EGFR pathway might be involved in this the pharmacologic inhibitor of EGFR, Iressa, or the vehicle, 2 sexual dimorphism. months after surgery. Because no differences were detected be- In conclusion, our study identifies a novel molecular path- tween Iressa- and vehicle-treated control mice, only one group is way critically involved in the genetic predisposition to develop ϫ shown. PAS staining, magnification, 200. (B and C): Proteinuria renal lesions after Nx. It also suggests that genes that encode for (B) and BP (C) in control (white bars) and Nx (black bars) mice molecules susceptible to modulate the activation of EGFR treated with either the vehicle or Iressa, 2 months after surgery. Data are means Ϯ SEM; n ϭ 6 for each experimental group. pathway could be putative modifiers of CKD progression. Statistical analysis: Nx versus control: *P Ͻ 0.05, **P Ͻ 0.01, More importantly, we have identified a novel therapeutic strat- ***P Ͻ 0.001; Iressa versus vehicle: ##P Ͻ 0.01 (ANOVA, followed egy to counteract the genetic predisposition to CKD progres- by Tukey-Kramer test). sion.

However, genetic variants in molecules involved in the post- transcriptional control of TGF-␣ expression could also act as CONCISE METHODS candidate modifiers. In fact, a complex network of proteins controls the amount of TGF-␣ that is biologically available by Animals modulating either the targeting of the precursor to the apical Mice used for these studies were FVB/N, C57BL/6, DBA/2, 129S2/Sv, membrane or its cleavage into the mature form.36,37 Interest- (C57BL/6x DBA/2)F1 (denoted B6D2F1) (Charles River), and ingly, we previously showed that angiotensin II causes TGF-␣ (C57BL/6xSJL)F1 (denoted B6SJLF1) (Janvier). The parental FVB/N overexpression by favoring the redistribution of its sheddase to and B6D2F1 strains were mated to produce the reciprocal (FVB/ the apical membrane.21 Although none of these genes have NxB6D2F1)G1 progeny. The G1 females were crossed to either been shown to modulate the evolution of CKD, further studies FVB/N or B6D2F1 males to obtain the G2 progeny. All experiments are required to determine whether one of them may account were performed on 9-week-old mice, fed ad libitum, and housed at for the increased susceptibility to develop renal lesions. constant ambient temperature in a 12-hour light cycle. Animal pro- Haplotype analysis is a powerful tool to identify potential cedures were conducted in accordance with French government pol- candidates in a chromosomal region. Hence, we applied this icies (Ministe`re de l’Agriculture). strategy to refine the region and identified genes that contain informative SNPs between FVB/N, C57BL/6, and DBA/2. We Experimental Protocol found 125,294 SNPs within the critical region, among which Mice were subjected to subtotal nephrectomy (Nx) which consists of 9668 are polymorphic between FVB/N and both C57BL/6 and the excision of the right kidney and the two poles of the left kidney to DBA/2. Of these SNPs, 3904 are located within 104 annotated achieve 75% reduction of total renal mass. Control mice were sub- genes. Thirteen genes harbor nonsynonymous coding poly- jected to a sham operation with decapsulation of both kidneys. After morphisms, among which 10 are expressed in adult mouse surgery, mice were fed a defined diet containing 30% casein and 0.5% kidney (Supplemental Table 1). Notably, one of these potential sodium. candidates, Tprkb, has been implicated in human CKD pro- Several groups of mice were investigated in complementary stud- gression by the genomewide association studies mentioned ies. For strain susceptibility studies, 18 and 32 females for each strain above. This gene encodes for the p53-related protein-kinase- were subjected to either sham operation or Nx, respectively. For renal

332 Journal of the American Society of Nephrology J Am Soc Nephrol 22: 327–335, 2011 www.jasn.org BASIC RESEARCH function and expression studies, additional 6 control and 6 Nx mice Linkage Analysis for each FVB/N and B6D2F1 strain were studied. For G1 progeny Because all C57BL/6, DBA/2, and B6D2F1 mice are resistant to lesion studies, 49 females and 40 males were subjected to Nx. For G2 progeny development after Nx, we hypothesized that all three strains are ho- studies, 12 and 25 males and 34 and 29 females from G1xFVB/N and mozygous for “resistant” alleles. We pursued the study with B6D2F1 G1xB6D2F1 crosses, respectively, were investigated after Nx. For mice and scored B6 and D2 alleles as non-FVB for linkage analysis. pharmacologic EGFR inhibition, 24 FVB/N mice were divided into 4 Linkage was evaluated in females and males from G1xFVB and groups of 6 mice: (i) sham operation ϩ EGFR inhibitor Iressa (100 G1xB6D2F1 crosses, respectively, with the assumption that, in these mg/kg per d by daily gavages; a gift from AstraZeneca); (ii) sham mice, a marker closely linked to the putative modifier should be (i) operation ϩ vehicle of Iressa (1% NaCl, 0.5% hydroxypropylmethyl- homozygous for the FVB alleles in lesion-prone females and heterozy- cellulose, 1% Tween); (iii) Nx ϩ Iressa; and (iv) Nx ϩ vehicle of gous in the resistant ones and, conversely, (ii) homozygous for non- Iressa. FVB alleles in resistant males and heterozygous in the lesion-prone Mice were sacrificed 2 months after surgery. In addition, for the ones. For each marker, linkage was estimated by the Fischer exact test strain susceptibility studies, mice were also sacrificed 4 and 6 months for the fit for the expected renal phenotype (lesions versus no lesions), after surgery, whereas for the TGF-␣ time course study mice were also using Prism Software. Linkage was considered significant when the P sacrificed at 4 and 6 weeks after surgery. BP was recorded 1 week values were Ͻ0.05. Additional genotyping was performed for the in- before sacrifice in awake mice (n ϭ 6 for each experimental group) for terval that showed linkage P Ͻ 0.05. The 95% confidence interval was 4 consecutive days, using tail-cuff plethysmography and PowerLab/ calculated on the basis of the Bayesian credible interval method (R/ 4SP software (AD Instruments). Urine samples were collected from QTL). the same animals, using metabolic cages during the 24 hours before sacrifice. At the time of sacrifice, the kidney was removed for mor- Gene Sequencing phologic, protein, and mRNA studies. In addition, on G2 progeny, TGF-␣ cDNA (primers in 5Ј and 3Ј UTR sequences) was sequenced in liver was removed for DNA extraction (genome scan). the three parental FVB/N, DBA/2, and C57BL/6 strain. To test the relative TGF-␣ allele expression, cDNA was reverse-transcribed from kidney RNA of selected G2 mice that brought heterozygous FVB/N Renal Function and C57BL/6 TGF-␣ alleles (n ϭ 8), then amplified using primers Proteinuria was measured using an Olympus multiparametric auto- designed around the 3-bp polyA insertion (detected by sequencing in analyzer (Instrumentation Laboratory). FVB/N and DBA/2 as compared with the C57BL/6 mouse strain), and quantified using an ABI PRISM 3100 genetic analyzer. Renal Morphology Kidneys were fixed in 4% paraformaldehyde and paraffin-embedded, Ribonuclease Protection Assay and Real-Time Reverse- and 4-␮m sections were stained with periodic acid–Schiff (PAS), Transcription PCR Masson trichrome, hematoxylin and eosin, or Picro-Sirius Red. A TGF-␣ mRNA levels were quantified by a ribonuclease protection pathologist examined and evaluated all the sections in a blinded fash- assay as described.35 GAPDH was used as the normalization control. ion. Twenty randomly selected microscopic fields of the cortex were studied (ϫ200). The severity of renal lesions was evaluated using a Immunochemical Analysis Nikon digital camera Dx/m/1200 and Lucia software (Laboratory Im- TGF-␣ immunostaining on mouse kidney was performed as de- 35 aging Ltd.), as described previously. scribed.35 The mean extent and intensity of TGF-␣ staining was quan- tified with a Nikon digital camera Dx/m/1200 and Lucia Software in 10 selected microscopic fields of the cortex (ϫ200). Genome Scan Genomic DNA was purified from mouse livers with Qiagen DNeasy kit. Sixty-four microsatellite markers were selected to span each au- Western Blot Analysis ␣ tosome and X chromosome with an average distance between mark- TGF- immunoblotting of mouse kidneys was performed as de- ␣ ers of 19.7 cM. The criterion for marker selection was their informa- scribed previously using a rabbit anti–TGF- antibody (Abcam) di- tiveness to differentiate FVB/N from C57BL/6 and DBA/2 alleles luted 1:500, followed by a donkey horseradish peroxidase-linked sec- (http://www.cidr.jhmi.edu/mouse). When the initial genome scan ondary antibody (Amersham Pharmacia, Biotech) diluted 1:5000.20 ␣ identified a single locus on chromosome 6, additional microsatellite Mouse monoclonal anti– -tubulin antibodies (Sigma-Aldrich) were ␣Ϫ/Ϫ markers or SNPs (http://www.informatics.jax.org) were performed used as controls. Protein extracts from kidneys of TGF- mice along the chromosome. Microsatellites were amplified by PCR on a were used to confirm antibody specificity. Biometra Uno II thermocycler. The PCR products were analyzed ei- ther on 3% agarose gel or by electrophoresis on 4% polyacrylamide, 7 Data and Statistical Analyses M urea sequencing gel on an ALF Pharmacia Sequencer, or on ABI Data are expressed as means Ϯ SEM. Differences between the exper- PRISM 3100 genetic analyzer. SNPs were amplified by PCR on an imental groups were evaluated using ANOVA, followed when signif- Eppendorf Mastercycler and analyzed on a Biotage pyrosequencer icant (P Ͻ 0.05) by the Tukey-Kramer test. When only two groups PSQ96MA. were compared, the Mann-Whitney test was used.

J Am Soc Nephrol 22: 327–335, 2011 TGF-␣ Is Required for CKD Progression 333 BASIC RESEARCH www.jasn.org

ACKNOWLEDGMENTS regression of renal lesions of chronic nephropathies and diabetes. J Clin Invest 116: 288–296, 2006 16. Esposito C, He CJ, Striker GE, Zalups RK, Striker LJ: Nature and We are grateful to Stephanie Le Corre, Martine Muffat-Joly, Sophie severity of the glomerular response to nephron reduction is strain- Berissi, and Charle`ne Blanchet. We thank Marco Pontoglio, Bernard dependent in mice. Am J Pathol 154: 891–897, 1999 Grandchamps, and Marie-Claire Gubler for critical advice. We are 17. Kren S, Hostetter TH: The course of the remnant kidney model in grateful to AstraZeneca for Iressa. This work was supported by IN- mice. Kidney Int 56: 333–337, 1999 SERM, Universite´Paris Descartes, AP-HP, Subvention AMGEM/So- 18. Ma LJ, Fogo AB: Model of robust induction of glomerulosclerosis in mice: Importance of genetic background. Kidney Int 64: 350–355, ciete´deNe´phrologie, AURA (Paris), Fondation de la Recherche 2003 Me´dicale (Grant DEQ20061107968). 19. 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Bochud M, Brown MJ, Caulfield MJ, Connell JM, Cook HT, Cotlarciuc 33. Gattone VH 2nd, Kuenstler KA, Lindemann GW, Lu X, Cowley BD Jr., I, Davey Smith G, de Silva R, Deng G, Devuyst O, Dikkeschei LD, Rankin CA, Calvet JP: Renal expression of a transforming growth Dimkovic N, Dockrell M, Dominiczak A, Ebrahim S, Eggermann T, factor-alpha transgene accelerates the progression of inherited, slowly Farrall M, Ferrucci L, Floege J, Forouhi NG, Gansevoort RT, Han X, progressive polycystic kidney disease in the mouse. J Lab Clin Med Hedblad B, Homan van der Heide JJ, Hepkema BG, Hernandez- 127: 214–222, 1996 Fuentes M, Hypponen E, Johnson T, de Jong PE, Kleefstra N, Lagou 34. Wong RW, Kwan RW, Mak PH, Mak KK, Sham MH, Chan SY: Overex- V, Lapsley M, Li Y, Loos RJ, Luan J, Luttropp K, Mare´chal C, Melander pression of epidermal growth factor induced hypospermatogenesis in O, Munroe PB, Nordfors L, Parsa A, Peltonen L, Penninx BW, Perucha transgenic mice. J Biol Chem 275: 18297–18301, 2000 E, Pouta A, Prokopenko I, Roderick PJ, Ruokonen A, Samani NJ, Sanna 35. Pillebout E, Weitzman JB, Burtin M, Martino C, Federici P, Yaniv M, S, Schalling M, Schlessinger D, Schlieper G, Seelen MA, Shuldiner AR, Friedlander G, Terzi F: JunD protects against chronic kidney disease Sjo¨gren M, Smit JH, Snieder H, Soranzo N, Spector TD, Stenvinkel P, by regulating paracrine mitogens. J Clin Invest 112: 843–852, 2003 Sternberg MJ, Swaminathan R, Tanaka T, Ubink-Veltmaat LJ, Uda M, 36. Sunnarborg SW, Hinkle CL, Stevenson M, Russell WE, Raska CS, Vollenweider P, Wallace C, Waterworth D, Zerres K, Waeber G, Ware- Peschon JJ, Castner BJ, Gerhart MJ, Paxton RJ, Black RA, Lee DC: ham NJ, Maxwell PH, McCarthy MI, Jarvelin MR, Mooser V, Abecasis Tumor necrosis factor-alpha converting enzyme (TACE) regulates epi- GR, Lightstone L, Scott J, Navis G, Elliott P, Kooner JS: Genetic loci dermal growth factor receptor ligand availability. J Biol Chem 277: influencing kidney function and chronic kidney disease. Nat Genet 42: 12838–12845, 2002 373–375, 2010 37. Fernandez-Larrea J, Merlos-Suarez A, Urena JM, Baselga J, Arribas J: 28. Osterholm AM, He B, Pitkaniemi J, Albinsson L, Berg T, Sarti C, A role for a PDZ protein in the early secretory pathway for the target- Tuomilehto J, Tryggvason K: Genome-wide scan for type 1 diabetic ing of proTGF-alpha to the cell surface. Mol Cell 3: 423–433, 1999 nephropathy in the Finnish population reveals suggestive linkage to a 38. Silbiger S, Neugarten J: Gender and human chronic renal disease. single locus on chromosome 3q. Kidney Int 71: 140–145, 2007 Gend Med 5 [Suppl A]: S3–S10, 2008 29. Rogus JJ, Poznik GD, Pezzolesi MG, Smiles AM, Dunn J, Walker W, Wanic K, 39. Neugarten J, Acharya A, Silbiger SR: Effect of gender on the progres- Moczulski D, Canani L, Araki S, Makita Y, Warram JH, Krolewski AS: High- sion of nondiabetic renal disease: A meta-analysis. J Am Soc Nephrol density single nucleotide polymorphism genome-wide linkage scan for sus- 11: 319–329, 2000 ceptibility genes for diabetic nephropathy in type 1 diabetes: Discordant 40. Yanes LL, Sartori-Valinotti JC, Reckelhoff JF: Sex steroids and renal dis- sibpair approach. Diabetes 57: 2519–2526, 2008 ease: Lessons from animal studies. Hypertension 51: 976–981, 2008 30. Karnib HH, Chua S, Gharavi AG: Genes for diabetic nephropathy: Sweet prospects on the horizon. Kidney Int 71: 94–96, 2007 31. Zeng F, Singh AB, Harris RC: The role of the EGF family of ligands and receptors in renal development, physiology and pathophysiology. Exp See related editorial, “Identification of a Major Chronic Renal Failure Suscepti- Cell Res 315: 602–610, 2009 bility Locus in Mice: Perhaps EGFR Determines What Happens to eGFR,” on pages 201–203 32. Lowden DA, Lindemann GW, Merlino G, Barash BD, Calvet JP, Gat- tone VH 2nd: Renal cysts in transgenic mice expressing transforming Supplemental information for this article is available online at http://www. growth factor-alpha. J Lab Clin Med 124: 386–394, 1994 jasn.org/.

J Am Soc Nephrol 22: 327–335, 2011 TGF-␣ Is Required for CKD Progression 335 Supplementary Table 1 : Genes in the Ckdp1 locus

Gene start Gene end MGI ID Gene Symbol Gene Name 52111712 116105285 MGI:2389093 Bmd8 bone mineral density 8 75494350 75494483 MGI:3054099 Bmd20 bone mineral density 20 75494350 94175949 MGI:3609662 Efw epididymal fat weight 75732352 75732750 MGI:3647168 Gm9001 predicted gene 9001 75920266 75920505 MGI:3642204 Gm10450 predicted gene 10450 76071621 76072891 MGI:3644799 Gm5995 predicted gene 5995 76234019 76234922 MGI:3782300 Gm4124 predicted gene 4124 76405212 76440490 MGI:3644002 Gm9007 predicted gene 9007 76446637 76447625 MGI:3644000 Gm9008 predicted gene 9008 76687091 76694251 MGI:3782303 Gm4127 predicted gene 4127 76832544 77795573 MGI:88275 Ctnna2 catenin (cadherin associated protein), alpha 2 77192711 77195511 MGI:2389173 Lrrtm1 leucine rich repeat transmembrane neuronal 1 78018562 78019581 MGI:3646936 Gm9018 predicted gene 9018 78165872 78167107 MGI:3644832 Gm5576 predicted gene 5576 78320879 78323460 MGI:97478 Reg3b regenerating islet-derived 3 beta 78325868 78328857 MGI:1353426 Reg3d regenerating islet-derived 3 delta 78330703 78333833 MGI:109408 Reg3a regenerating islet-derived 3 alpha 78355149 78358091 MGI:97896 Reg2 regenerating islet-derived 2 78375977 78378660 MGI:97895 Reg1 regenerating islet-derived 1 78416262 78418868 MGI:109406 Reg3g regenerating islet-derived 3 gamma 78430425 78433661 MGI:3648837 Gm6255 predicted gene 6255 78446750 78447446 MGI:3648423 Gm6072 predicted gene 6072 78997284 78998817 MGI:3646896 Gm5311 predicted gene 5311 79491203 79491959 MGI:3648928 Gm6261 predicted gene 6261 79876408 79973150 MGI:3782594 Gm4409 predicted gene 4409 79968871 80760137 MGI:2389180 Lrrtm4 leucine rich repeat transmembrane neuronal 4 80434732 80434899 MGI:3032855 Im4 immunoregulatory 4 80605023 80609097 MGI:3782593 Gm4408 predicted gene 4408 81243677 81244123 MGI:3782329 Gm4153 predicted gene 4153 81790668 81791356 MGI:3647948 Gm4874 predicted gene 4874 81846593 81846670 MGI:3619414 Mir468 microRNA 468 81850458 81860794 MGI:1921470 1700009C05Rik RIKEN cDNA 1700009C05 gene 81873663 81909909 MGI:2141656 AW146020 expressed sequence AW146020 81907845 81915952 MGI:1926274 Mrpl19 mitochondrial ribosomal protein L19 81991037 82043093 MGI:2385247 Fam176a family with sequence similarity 176, member A 82000447 82002575 MGI:3802115 Gm15864 predicted gene 15864 82192168 82192808 MGI:3648353 Gm6294 predicted gene 6294 82352469 82510092 MGI:98475 Tacr1 tachykinin receptor 1 82596695 82602923 MGI:1914229 Pole4 polymerase (DNA-directed), epsilon 4 (p12 subunit) 82675041 82724453 MGI:1315197 Hk2 hexokinase 2 82837254 82841167 MGI:3646278 Gm6312 predicted gene 6312 82861879 82889763 MGI:1340055 Sema4f sema domain, immunoglobulin domain (Ig), TM domain, and short cytoplasmic domain 82980928 82983465 MGI:893587 Dok1 docking protein 1 82984167 83002556 MGI:1337004 Loxl3 lysyl oxidase-like 3 83001260 83005267 MGI:1928676 Htra2 HtrA serine peptidase 2 83004690 83007674 MGI:107789 Aup1 ancient ubiquitous protein 1 83007838 83017204 MGI:2136388 Dqx1 DEAQ RNA-dependent ATPase 83018319 83020219 MGI:1350935 Tlx2 T-cell leukemia, homeobox 2 83028384 83030841 MGI:1917087 Pcgf1 polycomb group ring finger 1 83036359 83038237 MGI:1342288 Lbx2 ladybird homeobox homolog 2 (Drosophila) 83051611 83059933 MGI:3045292 Ccdc142 coiled-coil domain containing 142 83056602 83059048 MGI:3783052 Gm15605 predicted gene 15605 83057045 83059933 MGI:1915749 Mrpl53 mitochondrial ribosomal protein L53 83065490 83068892 MGI:1929872 Mogs mannosyl-oligosaccharide glucosidase 83069038 83071553 MGI:104710 Wbp1 WW domain binding protein 1 83071759 83075425 MGI:1917270 Ino80b INO80 complex subunit B 83085457 83102573 MGI:107371 Rtkn rhotekin 83099355 83106391 MGI:1922909 Wdr54 WD repeat domain 54 83106459 83112937 MGI:1919087 1700003E16Rik RIKEN cDNA 1700003E16 gene 83115914 83150111 MGI:107745 Dctn1 dynactin 1 83116146 83122727 MGI:3783068 Gm15624 predicted gene 15624 83169822 83254939 MGI:2443220 Slc4a5 solute carrier family 4, sodium bicarbonate cotransporter, member 5 83255685 83267600 MGI:1338850 Mthfd2 methylenetetrahydrofolate dehydrogenase, methenyltetrahydrofolate cyclohydrolase 83276010 83293770 MGI:2442631 Mobkl1b MOB1, Mps One Binder kinase activator-like 1B (yeast) 83299141 83310130 MGI:1925903 Bola3 bolA-like 3 (E. coli) 83318378 83329896 MGI:2446229 Tet3 tet oncogene family member 3 83348433 83391672 MGI:2444739 B430006D22Rik RIKEN cDNA B430006D22 gene 83391749 83398316 MGI:2444713 B230319C09Rik RIKEN cDNA B230319C09 gene 83421193 83421316 MGI:2661146 Ssta3 susceptibility to Salmonella typhimurium antigens 3 83430211 83456963 MGI:1351602 Dguok deoxyguanosine kinase 83462899 83486259 MGI:104589 Actg2 actin, gamma 2, smooth muscle, enteric 83493200 83522429 MGI:1917777 Stambp STAM binding protein 83550727 83551700 MGI:3644728 Gm7424 predicted gene 7424 83594536 83606181 MGI:1859834 Clec4f C-type lectin domain family 4, member f 83621209 83627851 MGI:2180021 Cd207 CD207 antigen 83642800 83662195 MGI:3583303 Vax2os2 Vax2 opposite strand transcript 2 83652120 83660926 MGI:3583301 Vax2os1 Vax2 opposite strand transcript 1 83661258 83688298 MGI:1346018 Vax2 ventral anterior homeobox containing gene 2 83663863 83664025 MGI:1932503 Bw18 body weight QTL 18 83693011 83708803 MGI:103285 Atp6v1b1 ATPase, H+ transporting, lysosomal V1 subunit B1 Gene start Gene end MGI ID Gene Symbol Gene Name 83711750 83712634 MGI:1915184 1700124L16Rik RIKEN cDNA 1700124L16 gene 83712638 83718320 MGI:1922555 Ankrd53 ankyrin repeat domain 53 83718875 83725806 MGI:1096575 Tex261 testis expressed gene 261 83742597 83743000 MGI:3644945 Gm7443 predicted gene 7443 83744976 83753414 MGI:1860418 Nagk N-acetylglucosamine kinase 83755395 83781735 MGI:2386865 Paip2b poly(A) binding protein interacting protein 2B 83791643 83792240 MGI:3643752 Gm5312 predicted gene 5312 83793590 83795091 MGI:3645065 Gm6342 predicted gene 6342 83824193 83825424 MGI:3779464 Gm5138 predicted gene 5138 83846952 83847318 MGI:3647000 Gm7490 predicted gene 7490 83864352 83865890 MGI:3780084 Gm9676 predicted gene 9676 83864384 83936863 MGI:1203484 Zfml zinc finger, matrin-like 83958584 84161054 MGI:1349385 Dysf dysferlin 83981568 83982665 MGI:3646544 Gm7498 predicted gene 7498 84051200 84056996 MGI:3705288 Gm15475 predicted gene 15475 84362936 84373832 MGI:3642570 Gm10445 predicted gene 10445 84485870 84486315 MGI:1925267 4930504D19Rik RIKEN cDNA 4930504D19 gene 84521938 84543740 MGI:2176159 Cyp26b1 cytochrome P450, family 26, subfamily b, polypeptide 1 84568481 85019507 MGI:1923164 Exoc6b exocyst complex component 6B 84762175 84763286 MGI:3643248 Gm7507 predicted gene 7507 85026186 85026713 MGI:894683 Npm3-ps1 nucleoplasmin 3, pseudogene 1 85061410 85076119 MGI:3647625 Gm5878 predicted gene 5878 85061679 85062598 MGI:3643297 Gm6384 predicted gene 6384 85083679 85087812 MGI:103078 Spr sepiapterin reductase 85084117 85084258 MGI:3642035 Gm10328 predicted gene 10328 85090499 85093643 MGI:3642569 Gm10444 predicted gene 10444 85095523 85097924 MGI:3644060 Gm7484 predicted gene 7484 85104999 85106228 MGI:1345187 Spr-ps1 sepiapterin reductase pseudogene 1 85137925 85154352 MGI:95387 Emx1 empty spiracles homolog 1 (Drosophila) 85163045 85283416 MGI:2137681 Sfxn5 sideroflexin 5 85284957 85324628 MGI:1098586 Rab11fip5 RAB11 family interacting protein 5 (class I) 85286286 85286367 MGI:3629673 Mir705 microRNA 705 85373923 85378264 MGI:3053002 Noto notochord homolog (Xenopus laevis) 85381983 85396429 MGI:108048 Smyd5 SET and MYND domain containing 5 85396804 85401964 MGI:1920577 1700040I03Rik RIKEN cDNA 1700040I03 gene 85402070 85418376 MGI:107184 Cct7 chaperonin containing Tcp1, subunit 7 (eta) 85419568 85452988 MGI:1261912 Fbxo41 F-box protein 41 85461116 85463536 MGI:99252 Egr4 early growth response 4 85537525 85652747 MGI:1934606 Alms1 Alstrom syndrome 1 homolog (human) 85656701 85663199 MGI:3782661 Gm4477 predicted gene 4477 85662819 85677743 MGI:3646394 Gm7510 predicted gene 7510 85695475 85707068 MGI:3782659 Gm4475 predicted gene 4475 85710673 85715738 MGI:2136449 Cml3 camello-like 3 85715359 85754486 MGI:3779593 Gm6413 predicted gene 6413 85758479 85759857 MGI:3779382 Gm11128 predicted gene 11128 85767214 85770937 MGI:1916299 Cml5 camello-like 5 85780382 85782076 MGI:1915646 Cml4 camello-like 4 85785612 85786921 MGI:3782668 Gm4484 predicted gene 4484 85793974 85794456 MGI:3704271 Gm9769 predicted gene 9769 85815416 85819131 MGI:2136446 Cml2 camello-like 2 85839318 85839958 MGI:3645978 Gm7528 predicted gene 7528 85849045 85854878 MGI:1922791 1700019G17Rik RIKEN cDNA 1700019G17 gene 85860151 85865620 MGI:1913366 Cml1 camello-like 1 85865732 85880278 MGI:1917036 Tprkb Tp53rk binding protein 85882551 85883343 MGI:3644831 Nat8b N-acetyltransferase 8B 85888208 85889214 MGI:3645071 Gm7542 predicted gene 7542 85892262 85911661 MGI:1919352 Dusp11 dual specificity phosphatase 11 (RNA/RNP complex 1-interacting) 85967185 85970990 MGI:1349421 Figla folliculogenesis specific basic helix-loop-helix 86028078 86069549 MGI:87919 Add2 adducin 2 (beta) 86145415 86225151 MGI:98724 Tgfa transforming growth factor alpha 86234881 86235888 MGI:3644882 Gm7545 predicted gene 7545 86236390 86237336 MGI:3782402 Gm4226 predicted gene 4226 86284025 86284234 MGI:3704272 Gm10443 predicted gene 10443 86315718 86320051 MGI:1913738 Fam136a family with sequence similarity 136, member A 86321546 86328896 MGI:1915261 Snrpg small nuclear ribonucleoprotein polypeptide G 86336000 86347144 MGI:1914131 Pcyox1 prenylcysteine oxidase 1 86354213 86383397 MGI:107914 Tia1 cytotoxic granule-associated RNA binding protein 1 86388368 86423494 MGI:2141787 C87436 expressed sequence C87436 86420799 86430824 MGI:2444132 A430078I02Rik RIKEN cDNA A430078I02 gene 86433370 86438221 MGI:1916897 2310040G24Rik RIKEN cDNA 2310040G24 gene 86474486 86476315 MGI:1345635 Pcbp1 poly(rC) binding protein 1 86578168 86579696 MGI:1915105 Asprv1 aspartic peptidase, retroviral-like 1 86600520 86619048 MGI:96908 Mxd1 MAX dimerization protein 1 86614289 86614572 MGI:3708740 Gm10442 predicted gene 10442 86625215 86634516 MGI:1913868 Snrnp27 small nuclear ribonucleoprotein 27 (U4/U6.U5) 86641762 86683372 MGI:1345156 Gmcl1 germ cell-less homolog 1 (Drosophila) 86686834 86715904 MGI:88030 Anxa4 annexin A4 86756799 86756921 MGI:3035966 Pbwg8 postnatal body weight growth 8 86798249 86799642 MGI:1914420 2610306M01Rik RIKEN cDNA 2610306M01 gene 86799511 86941858 MGI:1098687 Aak1 AP2 associated kinase 1 86959230 86978455 MGI:1913290 Nfu1 NFU1 iron-sulfur cluster scaffold homolog (S. cerevisiae) 86975513 86978396 MGI:1261789 D15Ertd556e DNA segment, Chr 15, ERATO Doi 556, expressed Gene start Gene end MGI ID Gene Symbol Gene Name 86992840 87042191 MGI:95698 Gfpt1 glutamine fructose-6-phosphate transaminase 1 87083848 87285714 MGI:1916788 Antxr1 anthrax toxin receptor 1 87295647 87300909 MGI:1913533 Gkn1 gastrokine 1 87323359 87329488 MGI:1913534 Gkn2 gastrokine 2 87333262 87338929 MGI:1916138 1190003M12Rik RIKEN cDNA 1190003M12 gene 87375892 87376028 MGI:2150764 Bits2 bitterness sensitivity 2 87375892 87376028 MGI:2662761 Egrm2 early growth rate, maternal effect 2 87375892 87376028 MGI:2680928 Eila2 ethanol induced locomotor activity 2 87375892 87376028 MGI:3639952 Lbn5 long bones 5 87375892 87376028 MGI:2680096 Rear1 rearing 1 87375892 87376028 MGI:2149052 Skl3 skeletal size (tail length) 3 87375892 87376028 MGI:1351483 Skts11 skin tumor susceptibility 11 87379027 87384486 MGI:1338820 Bmp10 bone morphogenetic protein 10 87408539 87483253 MGI:2443687 Arhgap25 Rho GTPase activating protein 25 87528586 87540695 MGI:1929676 Prokr1 prokineticin receptor 1 87578423 87622141 MGI:1919353 Aplf aprataxin and PNKP like factor 87622000 87672033 MGI:3586838 1810020O05Rik Riken cDNA 1810020O05 gene 87622376 87629280 MGI:2443140 E230015B07Rik RIKEN cDNA E230015B07 gene 87680863 87684011 MGI:3611451 Ccdc48 coiled-coil domain containing 48 87714987 87716198 MGI:3647624 Gm5879 predicted gene 5879 87728130 87729756 MGI:1860137 Gp9 glycoprotein 9 (platelet) 87738847 87762158 MGI:1917084 Rab43 RAB43, member RAS oncogene family 87768783 87788671 MGI:1923310 Isy1 ISY1 splicing factor homolog (S. cerevisiae) 87793076 87801100 MGI:88431 Cnbp cellular nucleic acid binding protein 87837808 87863589 MGI:1858696 Copg coatomer protein complex, subunit gamma 87863929 87886623 MGI:1914053 8430410A17Rik RIKEN cDNA 8430410A17 gene 87898448 87899446 MGI:3641929 Gm10249 predicted gene 10249 87930415 87931631 MGI:2685307 H1fx H1 histone family, member X 87930877 87955465 MGI:3648213 Gm5577 predicted gene 5577 87938324 87946457 MGI:3782427 Gm4250 predicted gene 4250 87949100 87995264 MGI:105068 Rab7 RAB7, member RAS oncogene family 88034514 88053950 MGI:98084 Rpn1 ribophorin I 88148328 88156483 MGI:95662 Gata2 GATA binding protein 2 88172262 88173251 MGI:1922801 Dnajb8 DnaJ (Hsp40) homolog, subfamily B, member 8 88172596 88175672 MGI:1920567 1700031F10Rik RIKEN cDNA 1700031F10 gene 88207865 88396507 MGI:2137092 Eefsec eukaryotic elongation factor, selenocysteine-tRNA-specific 88415403 88447566 MGI:1928760 Ruvbl1 RuvB-like protein 1 88453595 88468899 MGI:1858417 Sec61a1 Sec61 alpha 1 subunit (S. cerevisiae) 88497716 88577436 MGI:1918481 Klhdc6 kelch domain containing 6 88525054 88525172 MGI:3514274 Salpa2 serum alkaline phosphatase activity 2 88525054 88525172 MGI:2176909 Stheal5 soft tissue heal 5 88674406 88778354 MGI:1346042 Mgll monoglyceride lipase 88773355 88775755 MGI:106336 Mgll-rs2 monoglyceride lipase, related sequence 2 88785910 88791860 MGI:1933148 Abtb1 ankyrin repeat and BTB (POZ) domain containing 1 88792552 88825038 MGI:2442488 Podxl2 podocalyxin-like 2 88792848 88797268 MGI:3641714 Gm15612 predicted gene 15612 88832647 88833190 MGI:3782438 Gm4261 predicted gene 4261 88833469 88848694 MGI:105380 Mcm2 minichromosome maintenance deficient 2 mitotin (S. cerevisiae) 88852245 88862232 MGI:1345190 Tpra1 transmembrane protein, adipocyte asscociated 1 88931018 88931164 MGI:2668935 Fcsa1 femoral cross-sectional area 1 88931018 88931164 MGI:1345294 Pas1c pulmonary adenoma susceptibility 1c 89046104 89060024 MGI:3026922 4933427D06Rik RIKEN cDNA 4933427D06 gene 89090999 89096010 MGI:3584270 Gm1965 predicted gene 1965 89121868 89125522 MGI:3782444 Gm4267 predicted gene 4267 89126124 89131295 MGI:3782449 Gm4272 predicted gene 4272 89134574 89136019 MGI:3645279 Gm6507 predicted gene 6507 89143068 89144483 MGI:3645511 Gm6508 predicted gene 6508 89151075 89151554 MGI:3643385 Gm7679 predicted gene 7679 89154081 89155527 MGI:3645589 Gm6511 predicted gene 6511 89162240 89166073 MGI:2685685 Gm839 predicted gene 839 89219505 89235719 MGI:1922381 4930512J16Rik RIKEN cDNA 4930512J16 gene 89268851 89312592 MGI:107685 Plxna1 plexin A1 89333154 89545609 MGI:1913348 Chchd6 coiled-coil-helix-coiled-coil-helix domain containing 6 89593982 89625523 MGI:2386711 Txnrd3 thioredoxin reductase 3 89648651 89649480 MGI:2148531 V1r-ps1 vomeronasal 1 receptor, pseudogene 1 89664197 89665129 MGI:2148518 V1rb7 vomeronasal 1 receptor, B7 89696476 89697408 MGI:2148520 V1rb9 vomeronasal 1 receptor, B9 89739452 89740405 MGI:2148532 V1r-ps2 vomeronasal 1 receptor, pseudogene 2 89755002 89755938 MGI:2148533 V1r-ps3 vomeronasal 1 receptor, pseudogene 3 89794590 89795531 MGI:2148511 V1ra6 vomeronasal 1 receptor, A6 89819507 89820448 MGI:2148510 V1ra5 vomeronasal 1 receptor, A5 89843268 89844200 MGI:2148517 V1rb4 vomeronasal 1 receptor, B4 89857671 89858612 MGI:2148534 V1r-ps4 vomeronasal 1 receptor, pseudogene 4 89881644 89890501 MGI:1333762 V1ra2 vomeronasal 1 receptor, A2 89926165 89927094 MGI:2148519 V1rb8 vomeronasal 1 receptor, B8 89971882 89972814 MGI:2148509 V1ra4 vomeronasal 1 receptor, A4 89985927 89986835 MGI:2148508 V1ra3 vomeronasal 1 receptor, A3 90022080 90023012 MGI:1344384 V1rb2 vomeronasal 1, receptor B2 90057269 90058201 MGI:2148515 V1rb1 vomeronasal 1 receptor, B1 90078418 90080639 MGI:1333759 V1ra1 vomeronasal 1 receptor, A1 90103041 90103996 MGI:3646782 Gm6547 predicted gene 6547 90128710 90129639 MGI:2148512 V1ra7 vomeronasal 1 receptor, A7 Gene start Gene end MGI ID Gene Symbol Gene Name 90152811 90153650 MGI:2148513 V1ra8 vomeronasal 1 receptor, A8 90173402 90174334 MGI:2148516 V1rb3 vomeronasal 1 receptor, B3 90185659 90186267 MGI:3782465 Gm4287 predicted gene 4287 90192316 90193237 MGI:2148535 V1r-ps5 vomeronasal 1 receptor, pseudogene 5 90219100 90220047 MGI:2148514 V1ra9 vomeronasal 1 receptor, A9 90251264 90255442 MGI:2679261 BC048671 cDNA sequence BC048671 90259322 90275179 MGI:1919047 Chst13 carbohydrate (chondroitin 4) sulfotransferase 13 90283300 90314545 MGI:2385332 Uroc1 urocanase domain containing 1 90319486 90353484 MGI:1933108 Zxdc ZXD family zinc finger C 90353730 90378791 MGI:2141635 Ccdc37 coiled-coil domain containing 37 90412570 90425232 MGI:1929988 Klf15 Kruppel-like factor 15 90436421 90550197 MGI:1340024 Aldh1l1 aldehyde dehydrogenase 1 family, member L1 90546679 90549942 MGI:3783199 Gm15756 predicted gene 15756 90554719 90596406 MGI:1918949 Slc41a3 solute carrier family 41, member 3 90609592 90714135 MGI:1196356 Iqsec1 IQ motif and Sec7 domain 1 90794972 90795454 MGI:3648214 Gm16376 predicted gene 16376 90855813 90856447 MGI:3647847 Gm7749 predicted gene 7749 90963062 91066823 MGI:1859555 Nup210 nucleoporin 210 91106659 91124686 MGI:2385252 Hdac11 histone deacetylase 11 91108707 91109250 MGI:3705150 Gm14573 predicted gene 14573 91162449 91222534 MGI:95488 Fbln2 fibulin 2 91270515 91271029 MGI:3643336 Gm7755 predicted gene 7755 91313975 91361357 MGI:98961 Wnt7a wingless-related MMTV integration site 7A 91378799 91380099 MGI:3782758 Gm4575 predicted gene 4575 91386033 91396913 MGI:3779610 Gm6582 predicted gene 6582 91414271 91423417 MGI:1919420 Chchd4 coiled-coil-helix-coiled-coil-helix domain containing 4 91423697 91438453 MGI:1921372 Tmem43 transmembrane protein 43 91439299 91465878 MGI:103557 Xpc xeroderma pigmentosum, complementation group C 91465922 91472619 MGI:1914928 Lsm3 LSM3 homolog, U6 small nuclear RNA associated (S. cerevisiae) 91634089 91709055 MGI:98488 Slc6a6 solute carrier family 6 (neurotransmitter transporter, taurine), member 6 91711503 91746335 MGI:2681173 Grip2 glutamate receptor interacting protein 2 91828047 91849837 MGI:2444652 C130022K22Rik RIKEN cDNA C130022K22 gene 91854227 91900719 MGI:2685917 4930590J08Rik RIKEN cDNA 4930590J08 gene 91928872 92025998 MGI:2443369 Fgd5 FYVE, RhoGEF and PH domain containing 5 91962388 91966111 MGI:1923066 4930466I24Rik RIKEN cDNA 4930466I24 gene 92041384 92123051 MGI:1352466 Nr2c2 nuclear receptor subfamily 2, group C, member 2 92119519 92134027 MGI:1928140 Mrps25 mitochondrial ribosomal protein S25 92136706 92164919 MGI:1925537 Zfyve20 zinc finger, FYVE domain containing 20 92192055 92194644 MGI:98823 Trh thyrotropin releasing hormone 92230909 92231387 MGI:3643127 Gm7794 predicted gene 7794 92320902 92517360 MGI:1925144 Prickle2 prickle-like 2 (Drosophila) 92514423 92515834 MGI:3647623 Gm5880 predicted gene 5880 92666497 92666691 MGI:3664266 Dbm1 diabetes modifier 1 92722693 92893486 MGI:1916320 Adamts9 a disintegrin-like and metallopeptidase (reprolysin type) with thrombospondin type 1 motif, 9 92766472 92797168 MGI:1921766 A730049H05Rik RIKEN cDNA A730049H05 gene 92819351 92834405 MGI:3783179 Gm15737 predicted gene 15737 93086998 93087984 MGI:3646641 Gm5313 predicted gene 5313 93227374 93239567 MGI:3782499 Gm4318 predicted gene 4318 93413943 93414087 MGI:3046214 Pfat2 predicted fat percentage 2 93599341 93600219 MGI:3646293 Gm7812 predicted gene 7812 93628030 94233316 MGI:1203522 Magi1 membrane associated guanylate kinase, WW and PDZ domain containing 1 94273169 94308291 MGI:3782504 Gm4323 predicted gene 4323 94367794 94368238 MGI:3646507 Gm7825 predicted gene 7825 94450347 94553841 MGI:1914832 Slc25a26 solute carrier family 25 (mitochondrial carrier, phosphate carrier), member 26 94554523 94650152 MGI:107935 Lrig1 leucine-rich repeats and immunoglobulin-like domains 1 94672171 94672569 MGI:3779619 Gm6638 predicted gene 6638 94770524 94771513 MGI:3643837 Gm7833 predicted gene 7833 94798391 94798875 MGI:3782513 Gm4331 predicted gene 4331 94991160 94993382 MGI:3782769 Gm4586 predicted gene 4586 95064011 95067625 MGI:1919467 1700010K10Rik RIKEN cDNA 1700010K10 gene 95067234 95079712 MGI:2661430 Kbtbd8 kelch repeat and BTB (POZ) domain containing 8 95195739 95204552 MGI:3647796 Gm5723 predicted gene 5723 95226695 95283972 MGI:3041245 AY512915 cDNA sequence AY512915 95272106 95272204 MGI:3642207 Gm10234 predicted gene 10234 95424140 95668782 MGI:1306824 Suclg2 succinate-Coenzyme A ligase, GDP-forming, beta subunit 95875714 95876914 MGI:3644048 Gm7838 predicted gene 7838 95982854 95991694 MGI:3779532 Gm5881 predicted gene 5881 96063148 96607192 MGI:2443695 Fam19a1 family with sequence similarity 19, member A1 96066417 96067258 MGI:3782771 Gm4588 predicted gene 4588 96114491 96116237 MGI:1925732 1700123L14Rik RIKEN cDNA 1700123L14 gene 96605945 96606143 MGI:109357 Ots1 ovarian teratoma susceptibility 1 96793522 97010407 MGI:2444563 Fam19a4 family with sequence similarity 19, member A4 97060018 97099176 MGI:2141669 A130022J15Rik RIKEN cDNA A130022J15 gene 97105260 97129118 MGI:2684999 Tmf1 TATA element modulatory factor 1 97134022 97155337 MGI:1341217 Uba3 ubiquitin-like modifier activating enzyme 3 97160683 97183309 MGI:1929501 Arl6ip5 ADP-ribosylation factor-like 6 interacting protein 5 97188522 97202774 MGI:2444169 Lmod3 leiomodin 3 (fetal) 97236861 97567535 MGI:2141794 Frmd4b FERM domain containing 4B 97679023 97680012 MGI:3646971 Gm7320 predicted gene 7320 97757052 97971343 MGI:104554 Mitf microphthalmia-associated transcription factor 97840282 97840533 MGI:3782979 Gm15531 predicted gene 15531 98188071 98292666 MGI:2685611 Gm765 predicted gene 765 Gene start Gene end MGI ID Gene Symbol Gene Name 98196031 98196329 MGI:3708741 Gm10262 predicted gene 10262 98225530 98226110 MGI:3645233 Gm7892 predicted gene 7892 98457720 98647619 MGI:3779768 Gm7865 predicted gene 7865 98801357 98810809 MGI:3646989 Gm7875 predicted gene 7875 98875332 99216526 MGI:1914004 Foxp1 forkhead box P1 98943982 98944100 MGI:2150703 Obq14 obesity QTL 14 99098361 99098927 MGI:3779370 Gm11118 predicted gene 11118 99207478 99616791 MGI:1914142 Eif4e3 eukaryotic translation initiation factor 4E member 3 99642673 99643812 MGI:1202299 Gpr27 G protein-coupled receptor 27 99661344 99676347 MGI:1354178 Prok2 prokineticin 2 99720635 99721121 MGI:3646639 Gm5314 predicted gene 5314 99789637 99790316 MGI:3643996 Gm6681 predicted gene 6681 99827810 99828328 MGI:3650388 Gm14456 predicted gene 14456 100178559 100237352 MGI:1929059 Rybp RING1 and YY1 binding protein 100523077 100621151 MGI:1919421 Shq1 SHQ1 homolog (S. cerevisiae) 100554677 100555114 MGI:3644755 Gm6565 predicted gene 6565 100654728 100755075 MGI:2682940 Glt8d4 glycosyltransferase 8 domain containing 4 100676228 100676754 MGI:3783024 Gm15576 predicted gene 15576 100783632 100818711 MGI:3027896 Ppp4r2 protein phosphatase 4, regulatory subunit 2 100978016 101031199 MGI:3644643 Gm5882 predicted gene 5882 101033872 101034216 MGI:3782544 Gm4359 predicted gene 4359 101099603 101327891 MGI:1933157 Pdzrn3 PDZ domain containing RING finger 3 101488747 101490855 MGI:3641666 Gm10009 predicted gene 10009 101724243 101751976 MGI:3647145 Gm9871 predicted gene 9871 102012204 102022244 MGI:3649127 Gm7924 predicted gene 7924 102113300 102414661 MGI:99534 Cntn3 contactin 3 102124024 102127106 MGI:3643727 Gm7938 predicted gene 7938 102887347 102887532 MGI:3704274 Gm10082 predicted gene 10082 103460870 103683029 MGI:1098266 Chl1 cell adhesion molecule with homology to L1CAM 103500839 103503997 MGI:3642980 Gm7946 predicted gene 7946 103914203 103915630 MGI:3782563 Gm4378 predicted gene 4378 104442784 104813400 MGI:1858223 Cntn6 contactin 6 104453354 104453549 MGI:2448344 Hdlq11 HDL QTL 11 104453354 104453549 MGI:98773 Tmevd1 Theiler's murine encephalomyelitis virus induced demyelinating disease susceptibility 1 104680367 104681687 MGI:3779369 Gm11117 predicted gene 11117 105013248 105033785 MGI:3643497 Gm5315 predicted gene 5315 105481514 105481919 MGI:3782785 Gm4602 predicted gene 4602 105485938 105739128 MGI:3782564 Gm4379 predicted gene 4379 105624749 105627575 MGI:3783075 Gm15631 predicted gene 15631 105627654 106649304 MGI:1095737 Cntn4 contactin 4 105955399 105955597 MGI:2176188 Gasa3 gastritis type A susceptibility locus 3 106660378 106699031 MGI:96558 Il5ra interleukin 5 receptor, alpha 106719114 106732468 MGI:1917297 Trnt1 tRNA nucleotidyl transferase, CCA-adding, 1 106728243 106750068 MGI:1913277 Crbn cereblon 107043445 107044214 MGI:3645396 Gm6064 predicted gene 6064 107479777 107520204 MGI:106038 Lrrn1 leucine rich repeat protein 1, neuronal 107745604 107745840 MGI:1861748 Ichs immediate cutaneous hypersensitivity QTL 107745604 107745840 MGI:3531518 Obwq3 obesity and body weight QTL 3 108015039 108027116 MGI:1921979 Setmar SET domain and mariner transposase fusion gene 108057023 108135574 MGI:1889844 Sumf1 sulfatase modifying factor 1 108090107 108090513 MGI:3782787 Gm4604 predicted gene 4604 108163112 108501103 MGI:96623 Itpr1 inositol 1,4,5-triphosphate receptor 1 108421053 108421265 MGI:3642305 Gm10429 predicted gene 10429 108513599 108515582 MGI:3645626 Gm16433 predicted gene 16433 108610623 108616919 MGI:1097714 Bhlhe40 basic helix-loop-helix family, member e40 108733076 108773542 MGI:1914416 Arl8b ADP-ribosylation factor-like 8B 108778635 108809350 MGI:2180139 Edem1 ER degradation enhancer, mannosidase alpha-like 1 108831995 108832405 MGI:3648518 Gm6749 predicted gene 6749 109153975 109164855 MGI:3646196 Gm7261 predicted gene 7261 109393591 109394220 MGI:3782788 Gm4605 predicted gene 4605 110595862 111517224 MGI:1351344 Grm7 glutamate receptor, metabotropic 7 110909916 110910059 MGI:3055198 Tilss1 TNF-induced lethal shock susceptibility 1 111954645 111954732 MGI:2661704 Cfbw2 cystic fibrosis body weight 2 112160675 112162513 MGI:1914586 1700054K19Rik RIKEN cDNA 1700054K19 gene 112223752 112280419 MGI:1353635 Lmcd1 LIM and cysteine-rich domains 1 112236788 112238831 MGI:2442810 5031434C07Rik RIKEN cDNA 5031434C07 gene 112309318 112338017 MGI:2443733 D630042P16Rik RIKEN cDNA D630042P16 gene 112343293 112343811 MGI:3646736 Gm6118 predicted gene 6118 112409685 112422864 MGI:107570 Cav3 caveolin 3 112423678 112439802 MGI:109147 Oxtr oxytocin receptor 112513636 112515201 MGI:3782965 Gm15519 predicted gene 15519 112555538 112556773 MGI:3645010 Gm5578 predicted gene 5578 112569844 112646680 MGI:1890476 Rad18 RAD18 homolog (S. cerevisiae) 112667965 112897260 MGI:2152938 Srgap3 SLIT-ROBO Rho GTPase activating protein 3

The 10 genes, expressed in kidney, with known coding polymorphisms between FVB/N and both C57BL/6 and DBA/2 mice are highlighted in yellow.