BASIC RESEARCH www.jasn.org Maternal Environment Interacts with Modifier Genes to Influence Progression of Nephrotic Syndrome Julien Ratelade,*† Tiphaine Aguirre Lavin,*† Andrea Onetti Muda,*‡ Ludivine Morisset,*† ʈ ʈ Ge´raldine Mollet,*† Olivia Boyer,*†§ Deborah S. Chen,*† Anna Henger, Matthias Kretzler, Norbert Hubner,¶ Clotilde The´ry,** Marie-Claire Gubler,*† Xavier Montagutelli,†† Corinne Antignac,*†‡‡ and Ernie L. Esquivel*† *INSERM, U574, Hoˆpital Necker-Enfants Malades, †Universite´ Paris Descartes, Faculte´deMe´ decine Rene´ Descartes, Departments of §Pediatric Nephrology and ‡‡Genetics, Hoˆpital Necker-Enfants Malades, Assistance Publique- Hoˆpitaux de Paris, **INSERM, U653, Institut Curie, and ††Unite´deGe´ne´ tique des Mammife`res, Institut Pasteur, ʈ Paris, France; ‡Department of Pathology, Campus Biomedico University, Rome, Italy; Section of Nephrology, University of Michigan School of Medicine, Ann Arbor, Michigan; and ¶Max-Delbruck-Center for Molecular Medicine, Berlin-Buch, Germany ABSTRACT Mutations in the NPHS2 gene, which encodes podocin, are responsible for some cases of sporadic and familial autosomal recessive steroid-resistant nephrotic syndrome. Inter- and intrafamilial variability in the progression of renal disease among patients bearing NPHS2 mutations suggests a potential role for modifier genes. Using a mouse model in which the podocin gene is constitutively inactivated, we sought to identify genetic determinants of the development and progression of renal disease as a result of the nephrotic syndrome. We report that the evolution of renal disease as a result of nephrotic syndrome in Nphs2-null mice depends on genetic background. Furthermore, the maternal environment significantly interacts with genetic determinants to modify survival and progression of renal disease. Quantitative trait locus mapping suggested that these genetic determinants may be encoded for by genes on the distal end of chromosome 3, which are linked to proteinuria, and on the distal end of chromosome 7, which are linked to a composite trait of urea, creatinine, and potassium. These loci demonstrate epistatic interactions with other chromosomal regions, highlighting the complex genetics of renal disease pro- gression. In summary, constitutive inactivation of podocin models the complex interactions between maternal and genetically determined factors on the progression of renal disease as a result of nephrotic syndrome in mice. J Am Soc Nephrol 19: 1491–1499, 2008. doi: 10.1681/ASN.2007111268 Podocin is a 42-kD protein localized to the slit dia- groups.6–8 For instance, Ruf et al.9 described three phragms of terminally differentiated epithelial cells families in which sibling pairs bearing the same called podocytes,1 serving both structural and sig- podocin mutations had different ages of onset of naling functions.2,3 Mutations in the NPHS2 gene, encoding podocin, are responsible for 42% of fa- Received November 30, 2007. Accepted February 26, 2008. milial autosomal recessive and 10% of sporadic Published online ahead of print. Publication date available at cases of steroid-resistant nephrotic syndrome.4,5 Most www.jasn.org. patients present with nephrotic syndrome between 3 J.R. and T.A.L. contributed equally to this work. mo and 5 yr of age, rapidly progress to ESRD, and have Correspondence: 4 Dr. Ernie L. Esquivel, INSERM, U574, Hoˆpital no recurrence after transplantation. Necker-Enfants Malades, 149 rue de Se`vres, 75015 Paris, France. Interestingly, both inter- and intrafamilial vari- Phone: ϩ33-1-44-49-45-52; Fax: ϩ33-1-44-49-02-90; E-mail: ability in the renal disease among patients bearing [email protected] NPHS2 mutations have been reported by several Copyright ᮊ 2008 by the American Society of Nephrology J Am Soc Nephrol 19: 1491–1499, 2008 ISSN : 1046-6673/1908-1491 1491 BASIC RESEARCH www.jasn.org proteinuria and of ESRD. These reflect genetic modifiers and rable serum urea (Supplemental Figure S1) and light and elec- environmental factors influencing the rates of progression of tron microscopic findings (Supplemental Figures S2 and S3) in renal disease. In mice, inactivation of podocin leads to rapid the three strains. Foot processes were effaced in both immature development of diffuse mesangial sclerosis, massive protein- cortical and mature juxtamedullary glomeruli (Supplemental uria, and other sequelae of nephrotic syndrome, ultimately Figure S3); however, albuminuria levels, body weight reduc- leading to early, terminal renal failure.10 We previously showed tion, and decreases in nephrin and Trpc6 mRNA levels (Sup- that the survival of Nphs2 null mice is significantly longer in the plemental Figure S1) reflected more severe disease in B6 mice. 129S2/SvPas (129) strain, as compared with mice of mixed For observation of the evolution of renal disease, cohorts of C57BL/6 (B6):129 genetic background, consistent with genetic congenic FVB and 129 mice were killed at P5, P12, and P28. In modification.10 congenic FVB and 129 null mice at P5 and P12, mesangiolysis, The determinants of progression of renal disease in patients representing loss of mesangial cells or of the matrix they syn- with nephrotic syndrome are poorly understood. Mouse mod- thesize, was the predominant glomerular lesion (Figure 2 and els have proved invaluable in revealing genetic loci that affect Supplemental Figure S3). After quantification using a modi- renal disease severity in polycystic kidney disease,11,12 Alport fied mesangiolysis scoring system (Supplemental Figure S4), syndrome,13 and viral14 and toxic15 nephropathies. To under- FVB null mice had renal lesions of moderate (score 2.0) to stand better the genetic determinants of nephrotic syndrome, severe (score 3.0) mesangiolysis at P5, with a mean (Ϯ SEM) we characterized the renal phenotypes of podocin inactivation mesangiolysis score of 2.50 Ϯ 0.03 (Figure 2), reflecting 3.7% in three different mouse genetic backgrounds and carried out a of glomeruli having no (score 0) or mild (score 1) mesangial genetic modifier screen. Our data revealed that the maternal injury. In rare FVB null mice that survived to P12 (represent- lactational environment interacts with innate genetic determi- ing 10% of the cohort), the mean mesangiolysis score of 2.70 Ϯ nants elaborated by genes located within several modifier loci; 0.04 represented further worsening of glomerular lesions (Fig- hence, modeling combined gene–environment effects on renal ure 2). Moreover, FVB null mice at P5 had marked growth disease evolution. retardation, proteinuria, and an elevated plasma creatinine (56.2 Ϯ 5.5 versus 17.2 Ϯ 0.7 ␮mol/L in controls; P Ͻ 0.001), suggesting renal insufficiency (Supplemental Table S1). At RESULTS P12, plasma creatinine remained elevated (51.3 Ϯ 8.0 ␮mol/ L), but proteinuria could not be quantified because of paucity Early Survival and Evolution of Renal Disease Depend or absence of urine in the bladder. on Genetic Background We previously showed that Nphs2 inactivation resulted in an earlier demise of mixed B6:129 background mice than of con- genic 129 mice, suggesting a role for genetic modifiers.10 The null Nphs2 allele was, thus, additionally backcrossed onto glo- merulosclerosis-resistant B616 and glomerulosclerosis-sensi- tive FVB/N (FVB)17 strains, and the phenotypes of congenic null Nphs2 mice were characterized. Death occurred by post- natal (P) days P2, P5, and P18 on average in congenic B6, FVB, and 129 Nphs2 null mice, respectively (P Ͻ 0.0001 by log-rank test; Figure 1). Careful characterization at P1 showed compa- Figure 2. Evolution of renal disease in podocin null mice differs in the FVB and 129 strains. Severe mesangiolysis in both FVB and 129 null mice at P5 is followed by continued progression in the FVB null mice at P12 and, in contrast, by improvement in the 129 strain. At P28, a change from mesangiolysis to diffuse glomeru- Figure 1. Survival of Nphs2 null mice depends on genetic back- losclerosis in the 129 null mice is seen. FVB control mice (data not ground. Kaplan-Meier survival curve of cohorts of podocin null shown) have renal histology identical to 129 controls at each time mice in the B6, FVB, and 129 genetic backgrounds followed until point. Four-micron-thick sections were stained with periodic acid- the time of death. Schiff. Magnification, ϫ1000. 1492 Journal of the American Society of Nephrology J Am Soc Nephrol 19: 1491–1499, 2008 www.jasn.org BASIC RESEARCH A similar degree of mesangiolysis (score 2.50 Ϯ 0.04) was obtained via matings between FVB mothers and 129 fathers observed at P5 in 129 null mice (Figure 2). In contrast, the (F1-FVB) died on average at 8 d, whereas F1 null mice from mean mesangiolysis score decreased at P12 to 1.60 Ϯ 0.06 (P Ͻ 129 mothers and FVB fathers (F1-129) died at 22 d (log-rank 0.001), indicating significant improvement because 58% of test P Ͻ 0.0001). F1-FVB mice had significantly higher plasma glomeruli had mild or no mesangial injury (Figure 2). Ultra- urea, potassium (K), and proteinuria levels at P12 (Figure 3). structurally, foot processes at P12 remained diffusely effaced In addition, mesangiolysis was significantly more severe in F1- (data not shown). Subsequently, progressive glomerulosclero- FVB null mice than in F1-129 null mice, with mesangiolysis sis ensued, initially in a focal segmental and later in a diffuse scores of 2.90 Ϯ 0.02 and 2.00 Ϯ 0.05 (P Ͻ 0.001), respectively, pattern by P28 (Figure 2). Severe tubular dilation and atrophy corresponding to 0.67 versus 29.30% of glomeruli with no or and interstitial deposition of collagen were notable at this time mild lesions (Figure 4A). (data not shown). At P5, 129 null mice demonstrated body These differences between the F1 groups may result from weight loss (NS), proteinuria, and an elevated plasma creati- parent-of-origin effects as a result of genetic imprinting or gen- nine (35.1 Ϯ 2.2 versus 21.2 Ϯ 0.9 ␮mol/L in controls; P Ͻ der-linked or mitochondrial inheritance or from the maternal 0.001) but less than in FVB mice (Supplemental Table S1).