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1 Institute of Clinical Pharmacology and Toxicology
4 Department of Internal Medicine IV Nephrology, Universitätsklinikum Benjamin Franklin Hospital, Freie Universität Berlin, 12203 Berlin
2 Max-Delbrück Center for Molecular Medicine, Berlin Buch, 13125 Berlin, Germany
3 Department of Pathology, Leiden University Medical Center, 2300 RC, Leiden, The Netherlands
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ABSTRACT |
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genetics; linkage; quantitative trait loci
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INTRODUCTION |
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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.
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METHODS |
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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 F1 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 mm3 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.
F2 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-µm sections of the kidneys were stained with the periodic acid-Schiff technique. The glomerulosclerosis index (GSI) was assessed in selected F2 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 (http://www.rgd.mcw.edu/) and the Massachusetts Institute of Technology (http://www-genome.wi.mit.edu/rat/public/). 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.
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RESULTS |
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Cosegregation and QTL Mapping Analysis in F2 Animals
The overall range of UAE between 0.04 and 96.12 mg/24 h observed in F2 hybrids was in agreement with the contrasting data found between the two parental strains. However, only 5.75% (31 animals) of the 539 F2 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 F2 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 
1–1.5 mg/24 h in the F2 animals.
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DISCUSSION |
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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.
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 F1 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 F2 intercross (23, 25). The advantage of the F2 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 F1 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 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 F1 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 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 F2 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 F2 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 F2 rats, we could exclude structural differences by light microscopy in F2 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.
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DISCLOSURES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: R. Kreutz, Benjamin Franklin Klinikum, Freie Universität Berlin, Hindenburgdamm 30, 12203 Berlin, Germany (E-mail: Kreutz{at}medizin.fuberlin.de).
10.1152/physiolgenomics.00053.2003.
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