Early onset albuminuria in Dahl rats is a polygenetic trait that is independent from salt loading

Transcription

1 Physiol Genomics 14: , First published June 10, 2003; /physiolgenomics Early onset albuminuria in Dahl rats is a polygenetic trait that is independent from salt loading Ali Poyan Mehr, 1 Anja-Kristin Siegel, 1 Peter Kossmehl, 1 Angela Schulz, 1 Ralph Plehm, 2 Jan A. de Bruijn, 3 Emile de Heer, 4 and Reinhold Kreutz 1,4 1 Institute of Clinical Pharmacology and Toxicology, 4 Department of Internal Medicine IV Nephrology, Universitätsklinikum Benjamin Franklin Hospital, Freie Universität Berlin, Berlin; 2 Max-Delbrück Center for Molecular Medicine, Berlin Buch, Berlin, Germany; and 3 Department of Pathology, Leiden University Medical Center, 2300 RC, Leiden, The Netherlands Submitted 7 April 2003; accepted in final form 6 June 2003 Poyan Mehr, Ali, Anja-Kristin Siegel, Peter Kossmehl, Angela Schulz, Ralph Plehm, Jan A. de Bruijn, Emile de Heer, and Reinhold Kreutz. Early onset albuminuria in Dahl rats is a polygenetic trait that is independent from salt loading. Physiol Genomics 14: , First published June 10, 2003; /physiolgenomics The aim of the study was to characterize the genetic basis for the early onset of increased urinary albumin excretion (UAE) observed in the salt-sensitive Dahl rat (SS). We first characterized blood pressures and UAE values in adult SS compared with the spontaneously hypertensive rat (SHR) strain. Blood pressure measurements by radiotelemetry at 14 wk demonstrated similar spontaneous hypertension in both strains on a low-sodium diet containing 0.2% NaCl by weight, whereas UAE was markedly increased in SS compared with SHR ( vs mg/24 h, P ). Analysis of UAE in young animals of both strains fed a low-sodium diet demonstrated that UAE is elevated in SS as early as 4 wk of age (P ), when ultrastructural evaluation of glomeruli by electron microscopy appears still normal. At 8 wk SS demonstrated a 280-fold elevated UAE compared with SHR (P ). Consequently, to identify quantitative trait loci (QTLs) contributing to salt-independent early manifestation of increased UAE in the SS rat, we performed genome-wide linkage and QTL mapping analysis in a young F 2 population derived from the two contrasting strains. UAE was determined in 539 F 2 animals at 8 wk. We identified seven suggestive or significant UAE QTLs on rat chromosomes (RNO) RNO2, RNO6, RNO8, RNO9, RNO10, RNO11, and RNO19, accounting together for 34% of the overall variance of UAE in this F 2 population. Thus early onset albuminuria in the SS rat is under polygenetic influence and independent from salt loading. genetics; linkage; quantitative trait loci THE INBRED DAHL SALT-SENSITIVE rat strain SS was originally bred on the basis of its high blood pressure after being fed a high-salt (8% NaCl) diet (20). Thus this strain is generally used as a model for salt-sensitive hypertension by placing young animals on a high-salt Article published online before print. See web site for date of publication ( Address for reprint requests and other correspondence: R. Kreutz, Benjamin Franklin Klinikum, Freie Universität Berlin, Hindenburgdamm 30, Berlin, Germany ( Kreutz@medizin.fuberlin.de). diet, while blood pressures are determined some weeks thereafter. Consequently, it is usually disregarded that SS animals are also genetically prone to develop a spontaneous blood pressure increase under normal or low-sodium diet (13, 27). Moreover, Sterzel and associates (27) demonstrated previously that inbred SS animals derived from the SS/Jr strain inherit a glomerular phenotype leading to early manifestation of albuminuria prior to the consumption of a high-nacl diet and before the development of systemic hypertension. To characterize the genetic mechanisms leading to changes of the glomerular capillary wall filtration barrier giving rise to abnormal filtration of albumin and increased urinary albumin excretion (UAE) is of interest. Hence, an elevated UAE is a predictor for the development of chronic nephropathy (2) and a moderate increase of UAE in the range of microalbuminuria represents an independent risk factor for cardiovascular events in patients with hypertension, diabetes, or pre-existing cardiovascular disease (18, 22). We have recently demonstrated in the Munich- Wistar-Frömter (MWF) rat model the possibility to dissect the genetic basis of increased UAE by quantitative trait loci (QTL) mapping analysis (23). Accordingly, the goal of the current study was to characterize the genetic basis for the early onset of UAE observed in the SS rat. We characterized a spontaneously hypertensive rat (SHR) strain as a contrasting reference strain and showed similar hypertension on a low-sodium diet in this strain compared with SS. More importantly, this SHR strain shows contrasting low UAE rates compared with SS. We then performed an experimental cross-breeding study between SS and SHR for genome-wide QTL analysis and the genetic dissection of increased UAE in SS. METHODS Animals The SS rats are derived from the inbred SS/Jr strain available from Harlan Sprague-Dawley (Indianapolis, IN) (20). SS Fub animals and SHR Fub were obtained from our colonies (established in 1997) at the Freie Universität Berlin (FUB) and were maintained as reported (11). For the linkage analysis, we generated an (SS FUB SHR FUB)F 2 cross population including 539 male animals. Animals were studied in /03 $5.00 Copyright 2003 the American Physiological Society 209

2 210 GENETICS OF ALBUMINURIA IN DAHL RATS compliance with institutional regulations. Rats were grouped under conditions of regular 12-h diurnal cycles using an automated light switching device and climate-controlled conditions at a room temperature of 22 C. All parental and F 2 rats studied in the current set of experiments were fed a normal pelleted diet containing a low 0.2% NaCl by weight content and had free access to food and water. Determination of Phenotypes Blood pressure measurements. The development of spontaneous hypertension in both strains on low-sodium diet was studied by radiotelemetry (Data Sciences International, Minneapolis, MN) as reported (11). Transducers were implanted at 11 wk. Blood pressures were measured in freely moving conscious rats at 14 wk of age (n 6, respectively). Determination of UAE. For urine analysis, rats were placed into metabolic cages, and urine was collected over a 24-h period. Albumin concentrations were measured by a sensitive and rat-specific ELISA technique established in our laboratory (12) using a rat-specific antibody (ICN Biomedicals, Eschwege, Germany). UAE was measured in both parental strains including the animals evaluated by radiotelemetry (n 6, each), and in young animals studied in weekly intervals between 4 and 8 wk of age for time course analysis of UAE (n 11 12, each). In addition, UAE was determined in adult parental animals and in F 1 animals at 28 wk of age (n 11 12, each). Glomerular morphology analysis. For ultrastructural evaluation of glomeruli by electron microscopy, kidneys from parental animals at 4 wk (n 3 each) were evaluated. The kidneys were immersion-fixed with glutaraldehyde in 0.1 M cacodylate buffer. Small tissue pieces of cortex of 1 mm 3 were prepared using single-edge razorblades and subsequently embedded in Epon resin according to standard procedures (10). Ultrathin sections were cut using a Reichert-Jung ultramicrotome, contrasted with uranylcitrate, and subsequently analyzed in a Philips CM-10 transmission electron microscope. F 2 rats were killed under isoflurane anesthesia. The spleen, liver, and the left kidney were excised. For light microscopy evaluation, a midcoronal section of the left kidney was fixed in Methacarn and embedded in paraffin as previously described (24). The 3- m sections of the kidneys were stained with the periodic acid-schiff technique. The glomerulosclerosis index (GSI) was assessed in selected F 2 animals using a semiquantitative scoring system (21). Genotype Determination and QTL Mapping A complete genome screen on all 21 chromosomes except the Y chromosome was performed as reported (23). The interval between the 210 polymorphic microsatellite markers was on average 10 centimorgans (cm). The information and primer of microsatellites were obtained from data bases provided by the rat genome data base (RGD) at the Medical College of Wisconsin ( and the Massachusetts Institute of Technology ( Genotyping was performed as previously reported (11). QTL mapping analysis for UAE phenotypes was performed after logarithmic transformation of UAE values. Prior to linkage analysis, normal phenotypic distribution of UAE log values was confirmed by the Kolmogorov-Smirnov test. For the first genome screen 23 animals at the lowest and highest phenotype distribution for UAE were selected for analysis (total 46 animals), similar to a strategy recently reported (23, 25, 28). Subsequently, linkage analysis was performed with the MAPMAKER/EXP program to build a first genetic linkage map for this cross and to identify putative QTL by using the MAPMAKER/QTL computer package (15). For putative QTL regions, QTL mapping was performed after genetic analysis of all 539 animals for flanking and additional markers. QTL was considered to be significant if the logarithm of odds (LOD) score was more than 4.3 and suggestive if the LOD score was between 2.8 and 4.3 (14). Subsequently, we fixed the variance at each of the identified UAE QTLs with a LOD score higher than 2.8, respectively, and rescanned for each fixed QTL the total genome to test whether new potential QTL regions could be detected or whether a QTL with a suggestive LOD score achieved a significant LOD score after fixing. Statistical Analysis Statistical analysis was performed using one-way analysis of variance (ANOVA) or repeated measures analysis followed by the Bonferroni adjustment, and by Mann-Whitney U-test, as applicable. Data are means SD. Statistical significance was set at the P 0.05 level. RESULTS Phenotypes in Parental Strains Blood pressure measurements by radiotelemetry demonstrated under a low-salt diet that adult SS animals at the age of 14 wk develop increased blood pressure values similar to the SHR strain (Fig. 1A). In contrast, SS rats develop a marked increase in UAE compared with SHR animals (P , Fig. 1B). The time course of UAE in young animals (n 11 12) of both strains is shown in Fig. 2. As early as 4 wk of age, at the time of weaning, SS animals exhibited significantly elevated UAE rates, averaging mg/24 h compared with mg/24 h in SHR (P ). SS animals demonstrated a significant weekly increase of UAE between 4 wk and 8 wk of age (P 0.05), whereas SHR animals maintained low UAE rates below 1 mg/24 h. UAE averaged mg mg/24 h at 8 wk of age and was thus 280-fold elevated compared with SHR. With increasing age, adult SS animals at 28 wk of age had an UAE of mg/24 h, whereas SHR rats demonstrated mg/24 h (P ). At this age F 1 animals derived from both strains showed UAE values of mg/24 h, which were similar to those observed in SHR animals. Analysis of the glomerular ultrastructure by electron micrographs at 4 wk of age revealed no morphological abnormalities of glomeruli in both strains (Fig. 3). Podocytes still showed normal extensions, and the number of slit-pores had not decreased as yet in the SS strain. Neither in glomeruli from SHR rats nor from SS rats podocytes retraction or effacement was observed (Fig. 3). In addition, at 4 wk of age no morphological abnormalities were seen in SS animals by light microscopy evaluation (not shown).

3 GENETICS OF ALBUMINURIA IN DAHL RATS 211 Fig. 1. Blood pressure values (A) as determined by radiotelemetry in spontaneously hypertensive rats (SHR, white bars, n 6) and salt-sensitive Dahl rats (SS, black bars, n 6) and urinary albumin excretion (UAE, B) at 14 wk. Systolic and diastolic blood pressures (given as upper and lower confines of bars with standard deviation of the mean) are averaged over a 24-h period. Animals were fed a low-salt diet containing 0.2% NaCl by weight. Cosegregation and QTL Mapping Analysis in F 2 Animals The overall range of UAE between 0.04 and mg/24 h observed in F 2 hybrids was in agreement with the contrasting data found between the two parental strains. However, only 5.75% (31 animals) of the 539 F 2 hybrids showed an UAE rate higher than 1 mg/24 h, and only 2.41% (13 animals) demonstrated values above 4 mg/24 h. By genome-wide QTL mapping analysis we identified seven QTLs with either suggestive or significant linkage to UAE levels obtained in young F 2 animals at 8 wk of age (Fig. 4 and Table 1). Significant UAE QTLs (LOD 4.3) were mapped to rat chromosome (RNO) RNO2 with a peak LOD score of 4.4 at D2Rat126, RNO6 with a peak LOD score of 6.5 at D6Rat12, RNO9 with a peak LOD score of 8.0 at D9Rat10, and RNO19 with a peak LOD score of 5.5 at D19Rat75. Additional suggestive UAE QTLs (LOD between 2.8 and 4.3) were identified on RNO8 with a peak LOD score of 2.9 at D8Rat46, RNO10 with a peak LOD score of 3.6 at D10Rat30, and RNO11 with a peak LOD score of 3.7 at D11Mit2. When the variance at each UAE QTL was fixed and QTL mapping analysis was repeated, no additional suggestive QTLs were detected, and none of the identified suggestive QTLs achieved a significant LOD score. Taken together, the seven QTLs account for 34% of the overall variance of UAE in this population. The largest effect was contributed by the QTL on RNO9 showing the highest LOD score, i.e., 8.0, and contributing 8% to variance of the trait. When the phenotypic effect at each QTL was analyzed, it appeared that a significant albeit moderate increase of UAE in heterozygous animals occurred only at the QTL on RNO6, whereas a recessive mode of inheritance was evident at all other QTLs (Table 1). Overall, homozygosity at each UAE QTL accounted for a similar mean increase in UAE of mg/24 h in the F 2 animals. Interestingly, the presence of two SS alleles caused an increase of UAE at six QTLs as expected from the parental phenotypes. On RNO11, however, the presence of the SS allele was linked to lower UAE; thus homozygosity for the SHR allele led to a significant increase in UAE (Table 1). The functional importance of all interacting QTLs and thus of the suggestive QTL on RNO11, the two other suggestive QTLs (on RNO8 Fig. 2. Time course of UAE in SHR (open symbols, n 11) and SS (solid symbols, n 12). *P vs. SHR.

4 212 GENETICS OF ALBUMINURIA IN DAHL RATS Fig. 3. Ultrastructural morphology of the glomerulus of an SS rat (A and B) and an SHR rat (C and D) at4wkof age at low (A and C, 2,000) and high (B and D, 20,000 and 25,000, respectively) magnification. Note the virtual absence of podocyte retraction, effacement, and hypercellularity at this stage in the SS rat. and RNO10), and the four significant QTLs is supported by the finding of only one F 2 animal that carried two UAE increasing alleles at all seven QTLs including the two SHR alleles at the QTL on RNO11. This animal had an UAE rate of mg/24 h, while one F 2 rat that was homozygous for UAE increasing alleles at six QTLs (i.e., excluding RNO19) demonstrated a lesspronounced increase of UAE with mg/24 h (Fig. 5). Moreover, F 2 animals inheriting two UAE increasing alleles at five or less QTLs demonstrated a further decline in averaged UAE (Fig. 5). Glomerular morphology and GSI were determined by light microscopy in six F 2 animals demonstrating either high or low UAE rates in the total F 2 population, respectively. No difference in glomerular morphology between the two groups of F 2 animals was detected. DISCUSSION Recently, we have shown that the SHR strain used here as a contrasting reference strain for SS is capable of maintaining systolic blood pressure and UAE unchanged in response to a diet containing 4% NaCl by weight even after unilateral nephrectomy (21). In addition, no significant changes in glomerular structure and damage index were observed in SHR after unilateral nephrectomy and salt loading by light microscopy evaluation. These recent findings demonstrated that the SHR strain inherits a unique salt-resistant blood pressure phenotype and is at the same time resistant toward the development of functional and structural glomerular damage. Thus the SHR strain is well suited, even more so when studied under a low-sodium diet, as a contrasting reference strain for the SS rat with its distinct glomerular disease phenotype. This notion is supported by our time course analysis of UAE in young SHR and SS rats demonstrating a significant increase of UAE in SS as early as after weaning of the animals at 4 wk. This early contrasting phenotype led to a further 280-fold elevation compared with SHR at 8 wk, thus achieving a striking difference and setting the basis for cosegregation and linkage analysis. Our UAE data obtained in the SS strain at 4 wk are very similar to the findings reported by Sterzel et al. (27) more previously. In the latter study, however, SS animals were subsequently followed under a diet containing 0.9% NaCl. This could have contributed to an acceleration of both blood pressure development and renal damage with age in their longitudinal observation study due to the exquisite salt sensitivity of SS (27). It might have also affected the ultrastructural evaluation in this report, in which the authors observed in some glomeruli abnormalities of the glomerular basement barrier including thickening and broadening of podocyte foot process as important early signs of glomerular disease in somewhat older animals at 5 6 wk. In our study, in contrast, ultrastructural analysis by electron microscopy was carried out in younger animals at weaning at 4 wk and in animals that were raised together with their mothers on a diet containing a lower NaCl content (i.e., 0.2%). This evaluation demonstrated a normal glomerular structure including normal podocytes in SS. Thus, taken together, the current data clearly demonstrate that early onset of albuminuria in SS rats at 4 wk takes place before the onset of ultrastructural glomerular changes.

5 GENETICS OF ALBUMINURIA IN DAHL RATS 213 Fig. 4. Chromosomal maps and LOD plots for rat chromosomes (RNO) RNO2, RNO6, RNO9, and RNO19 demonstrating significant linkage (LOD 4.3) and RNO8, RNO10, and RNO11 demonstrating suggestive linkage (LOD between 2.8 and 4.3) to UAE. Chromosomal markers are given at the bottom of each chromosome and ordered according to the linkage map at distances of centimorgans (cm). A genetic distance of 10 cm is indicated. The horizontal dotted line at LOD 2.8 indicates the threshold for suggestive linkage, and the solid line at LOD 4.3 indicates the threshold for significant linkage. This is also observed in the hypertensive and albuminuric MWF rat in which morphological changes of the glomerular basement membrane or ultrastructure of the podocytes are not responsible for the increased UAE inherited in this strain (8). Therefore, genetic mechanisms leading to functional impairment of intrinsic properties of the glomerular wall rather than to structural impairment must be responsible for abnormal glomerular filtration of albumin and increased UAE in both the MWF strain (17) and the SS strain. Adult F 1 animals of the current cross between SS and SHR rats demonstrated UAE levels similar to those observed in the SHR strain, confirming an overall recessive genetic influence on UAE in SS animals. According to this finding, the conventional strategy would have been to perform a backcross study into the background of the disease model, i.e., SS, to increase the number of animals carrying two SS alleles at any given locus on average to 50% compared with 25% observed in an F 2 intercross (23, 25). The advantage of

6 214 GENETICS OF ALBUMINURIA IN DAHL RATS Table 1. Linkage to urinary albumin excretion in F 2 animals Locus Phenotype SS SR RR Statistics (ANOVA, P) Maximal LOD Score D2Rat (127) (267) (137) D6Rat (122) * (264) (145) D8Rat (136) (264) (126) D9Rat (129) (273) (133) D10Rat (105) (276) (132) D11Mit (128) (281) (106) D19Rat (125) (271) (137) Linkage results are presented for urinary albumin excretion (UAE). UAE phenotype values are means SD and are grouped according to genotypes; n values are in parentheses. SS, homozygous for the Dahl SS allele; SR, heterozygous animals carrying one SS allele and one allele of the spontaneously hypertensive (R) rat; RR, homozygous for the R allele. Maximal logarithm of odds (LOD) score values are given in when indicative for suggestive linkage (2.8 to 4.3) and are set in bold type when indicative for significant linkage ( 4.3). P values are given for one-way ANOVA carried out with UAE log values. *P 0.02 vs. RR genotype. the F 2 intercross performed here is to identify genotype-phenotype relations due to epistasis and genetic background that are not expected from phenotypes observed in parental strains or F 1 animals (7). A case in point represents the QTL on RNO11 showing suggestive linkage to UAE with the SHR allele causing an increase in UAE. This finding indicates the existence of a QTL protecting the SS strain from further increase in UAE. Resistance against the development of proteinuria has been also described in PVG/c rats (30) and in LEW/Moe rats (9) after induction of immune-mediated injury. Further research may provide new insights as to how the glomerulus is able to protect itself from loss of permselectivity in immune-meditated or non-immune-mediated injury. Alternatively, the SHR QTL locus may promote UAE, whereas this effect is normally masked in SHR parental animals due to compensation by the otherwise resistant genetic background. As evident from Table 1, the RNO11 QTL would not have been identified in a Fig. 5. UAE is shown at the vertical axis for 31 F 2 animals demonstrating a value of more than 1 mg/24 h. UAE values (solid square symbols) are grouped according to the bold numbers at the bottom of the horizontal axis, which indicate the number of quantitative trait loci carrying two UAE increasing alleles. The numbers in brackets indicate the number of animals in each group. UAE values were averaged when more than one animal contributed to a group. backcross population, which allows one only to compare animals with the SS and SR genotype. In addition, a trend toward higher UAE levels in heterozygous animals compared with animals carrying two RR alleles was observed at the QTLs on RNO8 and RNO19, and a significant albeit modest difference was detected on RNO6, which would have been missed in a backcross study as well. Nevertheless, the current result in F 1 animals demonstrating overall a recessive mode of inheritance for UAE in SS is in keeping with our data obtained in the MWF model, as ell as with results obtained by others in rat and mouse models in which the same inheritance pattern for renal disease phenotypes has been observed (16, 19, 25, 26). Of interest, a similar inheritance pattern seems to influence UAE also in diabetic and nondiabetic members of families with type 2 diabetes (2, 3). In a recent cross between MWF and normotensive Lewis reference rats we identified four suggestive or significant UAE QTLs on RNO1, RNO6, RNO12, and RNO17 by genome-wide QTL mapping (23). In the current study, we identified six UAE QTLs on different chromosomes, while the QTL on RNO6 identified in both crosses seem to overlap. Therefore, a different set of QTLs is involved in the polygenetic determination of UAE in the SS rat. Very recently, a backcross population between SS and SHR/NHsd rats, which were obtained from Harlan Sprague-Dawley, was also studied under a low-salt diet by Garrett et al. (4). In this report the authors performed a time course analysis of albuminuria and determined UAE at 8, 12, and 16 wk of age. They identified two UAE QTLs on RNO6 and eight additional QTLs on RNO1, RNO2, RNO8, RNO9, RNO10, RNO11, RNO13, and RNO19 (4). Most of these 10 QTLs were either present at week 8 and persisted through week 16 or became progressively more prominent over time. Moreover, the authors could show that most UAE QTLs colocalized with QTLs for structural renal damage as determined by light microscopy, whereas only the UAE QTL on RNO10 colocalized with a blood pressure QTL. Except the QTL2 on RNO6 and the QTLs on RNO1 and RNO13, the other seven UAE QTLs identified by Garrett et al. seem to overlap with

7 GENETICS OF ALBUMINURIA IN DAHL RATS 215 the QTLs identified in the current study (4). The fact that the two independent studies were performed in strains from different colonies and led largely to comparable results substantiates the relevance of the QTLs identified in both studies for the polygenetic determination of UAE in the SS rat. The necessity of synergistic interactions of multiple UAE QTLs for the manifestation of a considerable increase in UAE is highlighted in the current study by the two F 2 animals that inherited the homozygous state of two UAE increasing alleles at seven (i.e., all QTLs) or at six of the identified QTLs (Fig. 5). The genetics of salt-sensitive blood pressure in the SS model has been extensively studied, and 16 blood pressure QTLs have been identified in total (5). Recently, two studies for salt-sensitive blood pressure QTL mapping were performed between SS rats and models with spontaneous hypertension (5, 6). In a cross between SS and the albino surgery (AS), Garrett et al. (5) mapped blood pressure QTLs to RNO2, RNO4, and RNO8; in a cross between SS and SHR/NHsd rats, which were obtained from Harlan Sprague-Dawley, the authors mapped blood pressure QTLs to RNO3, RNO8, and RNO9 (6). In these experiments F 2 rats were exposed to a high-salt diet containing 8% NaCl, and data for renal damage or UAE were not reported. The QTLs for early onset albuminuria on RNO8 and RNO9 identified in the current study appear to colocalize with the previously identified blood pressure QTLs (5). Thus the existence of a common genetic basis for early onset glomerular dysfunction leading to increased UAE and salt-sensitive blood pressure increases in older animals appears possible and should be investigated in future studies. The aim of the current report was, however, to study the genetics of the glomerular phenotype leading to early onset of increase UAE in SS in the absence of structural glomerular changes. Sterzel et al. (27) demonstrated previously that SS animals develop progressive renal damage over time even when raised under a diet containing only 0.9% NaCl. Consequently, the genetic analysis of UAE in older animals is potentially confounded by structural glomerular changes, particularly when studied under higher salt diet. Nevertheless, the onset of increased UAE in young SS animals is not attributable to systemic hypertension or increases in glomerular capillary pressure as previously demonstrated (27). In addition, we have shown in the current study that onset of albuminuria is also not related to changes of glomerular ultrastructure, which is in agreement with other studies in animals and humans describing albuminuria in the absence of any ultrastructural alterations in the glomerular capillary wall (1, 29). Therefore, we set out to characterize the genetics of early onset albuminuria in the SS rat and could show that multiple QTLs are involved in this glomerular disease phenotype. Although we did not perform electron microscopy evaluation in the F 2 rats, we could exclude structural differences by light microscopy in F 2 animals with high UAE compared with those with low UAE. Our data provide the basis for further investigations to study the mechanisms leading to abnormal filtration of albumin across the glomerular capillary wall in the SS rat. We acknowledge the contributions of Reika Langanki, Petra Schwartz, and Simone Pechmann for excellent laboratory assistance, and the support of Frans Prins for transmission electron microscopy and Klaas van der Ham for digital image processing. DISCLOSURES This study was supported by grants from the Deutsche Forschungsgemeinschaft (DFG KR 1152/2-1, GK 462) and the Bundesministerium für Bildung und Forschung (BMBF Nationales Genomforschungsnetz: HK-Berlin 01GS0106/01GS0156). REFERENCES 1. Branten AJ, Van Den BJ, Jansen JL, Assmann KJ, and Wetzels JF. Familial nephropathy differing from minimal change nephropathy and focal glomerulosclerosis. Kidney Int 59: , Fogarty DG, Hanna LS, Wantman M, Warram JH, Krolewski AS, and Rich SS. Segregation analysis of urinary albumin excretion in families with type 2 diabetes. Diabetes 49: , Fogarty DG, Rich SS, Hanna L, Warram JH, and Krolewski AS. Urinary albumin excretion in families with type 2 diabetes is heritable and genetically correlated to blood pressure. Kidney Int 57: , Garrett MR, Dene H, and Rapp JP. Time-course genetic analysis of albuminuria in Dahl salt-sensitive rats on low-salt diet. J Am Soc Nephrol 14: , Garrett MR, Joe B, Dene H, and Rapp JP. Identification of blood pressure quantitative trait loci that differentiate two hypertensive strains. J Hypertens 20: , Garrett MR, Saad Y, Dene H, and Rapp JP. Blood pressure QTL that differentiate Dahl salt-sensitive and spontaneously hypertensive rats. Physiol Genomics 3: 33 38, Hilbert P, Lindpaintner K, Beckmann JS, Serikawa T, Soubrier F, Dubay C, Cartwright P, de Gouyon B, Julier C, Takahasi S, Vincent M, Ganten D, Georges M, and Lathrop GM. Chromosomal mapping of two genetic loci associated with blood-pressure regulation in hereditary hypertensive rats. Nature 353: , Iordache BE, Imberti O, Foglieni C, Remuzzi G, Bertani T, and Remuzzi A. Effects of angiotensin-converting enzyme inhibition on glomerular capillary wall ultrastructure in MWF/ Ztm rats. J Am Soc Nephrol 5: , Ketteler M, Westenfeld R, Gawlik A, Bachmann S, Frey A, Schonfelder G, Paul M, Distler A, and de Heer E. Nitric oxide synthase isoform expression in acute versus chronic anti- Thy 1 nephritis. Kidney Int 61: , Kootstra CJ, Bergijk EC, Veninga A, Prins FA, de Heer E, Abrahamson DR, and Bruijn JA. Qualitative alterations in laminin expression in experimental lupus nephritis. Am J Pathol 147: , Kreutz R, Hübner N, James MR, Bihoreau MT, Gaugier D, Lathrop GM, Ganten D, and Lindpaintner K. Dissection of a quantitative trait locus for genetic hypertension on rat chromosome 10. Proc Natl Acad Sci USA 92: , Kreutz R, Kovacevic L, Schulz A, Rothermund L, Ketteler M, and Paul M. Effect of high NaCl diet on spontaneous hypertension in a genetic rat model with reduced nephron number. J Hypertens 18: , Kurtz TW and Morris RCJ. Hypertension in the recently weaned Dahl salt-sensitive rat despite a diet deficient in sodium chloride. Science 230: , Lander E and Kruglyak L. Genetic dissection of complex traits: guidelines for interpreting and reporting linkage results. Nat Genet 11: , Lander ES, Green P, Abrahamson J, Barlow A, Daly MJ, Lincoln SE, and Newburg L. MAPMAKER: an interactive computer package for constructing primary genetic linkage

8 216 GENETICS OF ALBUMINURIA IN DAHL RATS maps of experimental and natural populations. Genomics 1: , Lenz O, Zheng F, Vilar J, Doublier S, Lupia E, Schwedler S, Striker LJ, and Striker GE. The inheritance of glomerulosclerosis in mice is controlled by multiple quantitative trait loci. Nephrol Dial Transplant 13: , Macconi D, Ghilardi M, Bonassi ME, Mohamed EI, Abbate M, Colombi F, Remuzzi G, and Remuzzi A. Effect of angiotensin-converting enzyme inhibition on glomerular basement membrane permeability and distribution of zonula occludens-1 in MWF rats. J Am Soc Nephrol 11: , Mann JF, Gerstein HC, Pogue J, Bosch J, and Yusuf S. Renal insufficiency as a predictor of cardiovascular outcomes and the impact of ramipril: the HOPE randomized trial. Ann Intern Med 134: , Murayama S, Yagyu S, Higo K, Ye C, Mizuno T, Oyabu A, Ito M, Morita H, Maeda K, Serikawa T, and Matsuyama M. A genetic locus susceptible to the overt proteinuria in BUF/Mna rat. Mamm Genome 9: , Rapp JP. Genetic analysis of inherited hypertension in the rat. Physiol Rev 80: , Rothermund L, Luckert S, Kossmehl P, Paul M, and Kreutz R. Renal endothelin ET(A)/ET(B) receptor imbalance differentiates salt-sensitive from salt-resistant spontaneous hypertension. Hypertension 37: , Ruilope LM, Salvetti A, Jamerson K, Hansson L, Warnold I, Wedel H, and Zanchetti A. Renal function and intensive lowering of blood pressure in hypertensive participants of the hypertension optimal treatment (HOT) study. J Am Soc Nephrol 12: , Schulz A, Litfin A, Kossmehl P, and Kreutz R. Genetic dissection of increased urinary albumin excretion in the Munich Wistar Fromter rat. J Am Soc Nephrol 13: , Shibutani M and Uneyama C. Methacarn: a fixation tool for multipurpose genetic analysis from paraffin-embedded tissues. Methods Enzymol 356: , Shiozawa M, Provoost AP, van Dokkum RP, Majewski RR, and Jacob HJ. Evidence of gene-gene interactions in the genetic susceptibility to renal impairment after unilateral nephrectomy. J Am Soc Nephrol 11: , Stella P, Cusi D, Duzzi L, and Bianchi G. Relations between hypertension and glomerulosclerosis in first-generation hybrid rats of the Milan strains. Child Nephrol Urol 11: 6 9, Sterzel RB, Luft FC, Gao Y, Schnermann J, Briggs JP, Ganten D, Waldherr R, Schnabel E, and Kriz W. Renal disease and the development of hypertension in salt-sensitive Dahl rats. Kidney Int 33: , Stoll M, Kwitek-Black AE, Cowley AW Jr, Harris EL, Harrap SB, Krieger JE, Printz MP, Provoost AP, Sassard J, and Jacob HJ. New target regions for human hypertension via comparative genomics. Genome Res 10: , Topham PS, Kawachi H, Haydar SA, Chugh S, Addona TA, Charron KB, Holzman LB, Shia M, Shimizu F, and Salant DJ. Nephritogenic mab is directed at the extracellular domain of rat nephrin. J Clin Invest 104: , Van Dixhoorn MG, Salazar-Exaire D, Sato T, Daha MR, Quigg RJ, Bruijn JA, Couser WG, and de Heer E. Antivitronectin antibodies enhance anti-thy-1-induced proteinuria in PVG/c, but not in Wistar rats. J Am Soc Nephrol 9: , 1998.