Journal of the American Society of Nephrology
2007 JASN IMPACT FACTOR 7.111 HOME   AUTHOR INFO   EDITORIAL BOARD   SUBSCRIBE   FEEDBACK   ALERTS   HELP 
    advanced
CURRENT ISSUE ARCHIVES JASN Express ONLINE SUBMISSION


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Schulz, A.
Right arrow Articles by Kreutz, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schulz, A.
Right arrow Articles by Kreutz, R.
J Am Soc Nephrol 14:3081-3089, 2003
© 2003 American Society of Nephrology

BASIC SCIENCE

A Major Gene Locus Links Early Onset Albuminuria with Renal Interstitial Fibrosis in the MWF Rat with Polygenetic Albuminuria

Angela Schulz*, Dorothea Standke*, Larisa Kovacevic*, Martin Mostler*, Peter Kossmehl*, Monika Stoll{dagger} and Reinhold Kreutz*,{ddagger}

*Institute of Clinical Pharmacology and Toxicology and {ddagger}Department of Nephrology, Campus Benjamin Franklin, Charité–Universitätsmedizin Berlin, Berlin, Germany; and {dagger}Institute for Arteriosclerosis Research, Westfälische-Wilhelms-Universität Münster, Münster, Germany

Correspondence to Dr. Reinhold Kreutz, Charité–Universitätsmedizin Berlin, Campus Benjamin Franklin, Freie Universität Berlin, Hindenburgdamm 30, 12200 Berlin, Germany. Phone: +49-30-8445-2280; Fax: +49-30-8445-4482; E-mail: kreutz{at}medizin.fu-berlin.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. The development of renal interstitial fibrosis (RIF) represents an important step in the progression of chronic proteinuric nephropathies. The Munich Wistar Frömter (MWF) rat represents a valuable model to study the progression in proteinuric renal disease. MWF animals demonstrate a significant increase of urinary albumin excretion (UAE) and RIF compared with the spontaneously hypertensive rat (SHR) with low UAE. The aim of this study was to analyze the genetic basis and the relation between UAE and RIF by genetic linkage and quantitative trait loci (QTL) mapping analysis. The authors generated a backcross population between MWF and SHR including 215 male animals. UAE was determined in young backcross animals at 8 wk, and at 14 and 24 wk of age, respectively. RIF was evaluated by Sirius red staining of kidney sections and quantified by computer-assisted image analysis at 24 wk. Total genome scan analysis identified in total eight QTL linked to UAE and a major locus on chromosome 6. At this locus, homozygosity for the MWF allele exhibited a strong effect on UAE levels (threefold elevation) and displayed significant linkage already at 8 wk (logarithm of odds [LOD] = 4.3) with increasing significance at 14 and 24 wk (LOD = 7.8 and 10.1, respectively). In addition, this was the only QTL that was linked to the amount of RIF (P = 0.0009, LOD = 2.4). These data establish a genetic link between early onset albuminuria and progression of RIF at the QTL on RNO6. This study demonstrates the power of genetic linkage analysis for the dissection of physiologic pathways involved in renal disease progression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The Munich Wistar Frömter (MWF) rat represents a valuable experimental model for the investigation of progressive glomerular injury and the progression of proteinuric renal disease (1,2). Previous investigations in this model demonstrated that molecular mechanisms leading to functional impairment of intrinsic properties of the glomerular wall rather than structural changes must be responsible for abnormal permeability of the glomerular capillary barrier to proteins (1,3). This results in an early increase of urinary albumin excretion (UAE) in young MWF animals (4). A large body of experimental evidence obtained in MWF and other animal models of proteinuric renal disease documented a pathophysiologic link among progressive proteinuria, tubulointerstitial damage, and renal scarring (5). The data indicate that progression of chronic proteinuric nephropathies to end-stage renal disease follows a final common pathway despite the heterogenous nature of the initial insults in the glomerulus (6). The development of tubulointerstitial damage and renal interstitial fibrosis (RIF) are important steps in this pathway, and RIF may correlate with disease prognosis and organ survival (7–9). The pivotal first step, however, is the impairment of glomerular permeability to proteins with subsequent excessive filtration and presentation of these macromolecules to the proximal tubule. The progressive accumulation of RIF is a detrimental long-term result as a consequence of a complex tubulointerstitial damage response that is initiated by the increased protein filtration (5, 7). In this regard, the MWF model represents a unique model to investigate the genetic basis of both the molecular changes of the glomerular capillary wall filtration barrier, i.e., the pivotal first step, and the subsequent pathways of disease progression including the development of RIF.

We have recently demonstrated in the MWF model the possibility to dissect the genetic basis of increased UAE by quantitative trait loci (QTL) mapping analysis and demonstrated the polygenetic determination of increased UAE (4). The aim of the current report was to further analyze the genetic basis for the early onset of UAE in the MWF rat and to investigate the potential link between the development of UAE and RIF in an appropriate experimental setting. To this end—and in contrast to our recent study in which we used the normotensive Lewis rat as an experimental counterpart with low UAE (4)—the MWF strain was studied in an experimental backcross experiment with a spontaneously hypertensive rat (SHR) strain as a contrasting model. SHR show similar hypertension compared with MWF; more importantly, this strain shows contrasting low UAE rates (10) and significantly less RIF compared with MWF. We therefore performed an experimental crossbreeding study between MWF and SHR for genome-wide QTL analysis to analyze the genetic basis of UAE and RIF in the background of two strains with similar arterial hypertension.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
MWF/Fub and SHR/Fub rats were obtained from our colonies at the Freie Universität (FU) Berlin, Benjamin Franklin Campus, Germany, as reported (4,10). Animals were studied in 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. The rats were fed a normal pelleted diet containing 0.2% NaCl and had free access to food and water. For the genetic linkage analysis, we generated a (MWF x SHR) F1 x MWF backcross population including 215 male animals.

Phenotyping
In the parental MWF and SHR rats (n = 8 each) systolic BP (SBP) and UAE were measured at 14 wk of age, respectively. To account for age of onset effect in the backcross population, UAE and total urinary protein excretion (UPE) were measured in young animals at 8 wk of age and in adult animals at 14 and 24 wk, respectively. SBP was measured at 14 wk of age by a noninvasive tail-cuff method in awake animals using a computer-assisted oscillatory detection device (TSE, Bad Homburg, Germany) as described previously (11). Two training periods were performed on two separate days. The final BP measurements were subsequently recorded on the three consecutive days. Due to three sets of two measurements at each session, the individual BP phenotype that was accepted for linkage analysis was based on a maximum of 18 and a minimum of 12 measurements for each rat (11).

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 (11) using a rat-specific antibody (ICN Biomedicals, Eschwege, Germany). UPE was determined by the Bradford method. Plasma glucose and serum creatinine were measured with standard methods. Parental rats were subsequently sacrificed under ether anesthesia at 18 wk and backcross animals at 24 wk of age, respectively. The spleen and both kidneys were excised. The body and total kidney weights were determined. For light microscopy evaluation, a mid-coronal section of the left kidney was immersed in Dubosq-Brasil solution and embedded in paraffin for histologic studies. The 3-µm sections of the kidneys were stained with the periodic acid-Schiff technique (PAS) for the determination of glomerulosclerosis index (GSI) by using a semiquantitative scoring system analyzing both superficial and juxtamedullary glomeruli (12).

RIF was determined after staining of sections with Sirius red following previous recommendations (8). Quantification was performed with use of a video camera combined with a video control system (Sony MC-3255, AVT-horn GmbH) adapted to a Zeiss Axiophot microscope. Image analysis was performed with the use of freely available software (Scion Image 1.62a, Scion Co) on a Power Macintosh 8200/120 computer. After digitalization, gray-scale images were transformed into binary images, and the relation of Sirius red–stained interstitial area to total area of image was determined; ten sections per animal were averaged to obtain individual RIF phenotypes for each rat.

Two phenotypes for the presence of superficial glomeruli were determined: first, the number of surface glomeruli per section with direct contact to the surface was counted; second, all superficial glomeruli present in the renal cortex corticis zone but without direct surface contact were counted in three mid-coronal sections for each animal, respectively (4).

Genotype Determination and QTL Mapping
In the backcross population, a complete genome screen on all chromosomes except the Y-chromosome was performed. The interval between the polymorphic simple sequence length polymorphism (SSLP) markers was on average 10 centiMorgans (cM). The information and primer sequences of SSLP markers were obtained from databases provided by the rat genome database (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 by amplification of SSLP markers by PCR as described previously (13). The forward primer was labeled with [{gamma} 32P] ATP by T4 polynucleotide kinase. PCR products were processed and subsequently analyzed by autoradiography after polyacrylamide gel electrophoresis. All genotypes were independently analyzed by two investigators (AS and RK). After linkage analysis, genotypes were ordered along chromosomes in computer spreadsheets and checked for potential errors, e.g., unlikely double crossovers in relatively small regions, by visual inspection after color-coding and by the error-checking module in the MAPMAKER program. Linkage analysis was performed as previously reported (4) with the MAPMAKER/EXP and MAPMAKER/QTL 3.0b programs (14). A QTL was considered to be significant if the logarithm of odds (LOD) score was more than 3.3 and suggestive if the LOD score was between 1.9 and 3.3 (15).

Statistical Analyses
Statistical analyses were performed using ANOVA followed by Bonferroni adjustment and by Mann-Whitney U-test and Kruskal-Wallis test, as appropriate. Analysis of correlation was performed using the Pearson coefficient. Data are presented as means ± SD.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of Parental Strains
The data for body weight, kidney weight, serum creatinine, and plasma glucose are presented in Table 1. In MWF rats, the body weight, total kidney weight, and kidney weight to body weight ratio were significantly lower compared with SHR rats. Plasma glucose and serum creatinine levels exhibited no significant differences between both strains.


View this table:
[in this window]
[in a new window]

  		Table 1. Phenotypes for parental MWF and SHR animals
 
In MWF rats, SBP was comparable to SHR rats (Figure 1A). MWF rats showed UAE levels that were about 80 times higher than in SHR (Figure 1B, P < 0.0001). In contrast, F1-animals demonstrated mean UAE values of 0.35 ± 0.33 mg/24 h that were similar to those obtained in the parental SHR strain at 14 wk of age (0.62 ± 0.13 mg/24 h). The data for UPE were in accordance with the results obtained for UAE (Figure 1C).



View larger version (9K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 1. (A) Systolic BP (SBP), (B) urinary albumin excretion (UAE), and (C) urinary protein excretion (UPE) of parental male Munich Wistar Frömter (MWF) rats and spontaneously hypertensive rats (SHR) at 14 wk of age. * P < 0.01 compared with SHR.

 
Renal histology phenotypes in parental strains are presented in Table 2. The number of superficial glomeruli is significantly increased in MWF animals compared with SHR, and surface glomeruli were only detected in MWF as expected. GSI was moderately (1.6-fold) increased in MWF compared with SHR, whereas RIF (Figure 2, A and B) showed a more pronounced 2.7-fold elevation in MWF compared with SHR (P < 0.05, respectively, Table 2).


View this table:
[in this window]
[in a new window]

  		Table 2. Renal histology phenotypes in parental MWF and SHR animals
 


View larger version (106K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 2. Sirius red staining for renal interstitial fibrosis (RIF) of a representative parental MWF (RIF = 5.2%, panel A) and SHR (RIF = 1.9%, panel B) animal compared with a backcross animal with either a high (2.84%, panel C) or low (0.64%, panel D) RIF value.

 
Cosegregation and Linkage Analysis in Backcross Animals
Descriptive statistics for UAE and UPE phenotypes of backcross animals are summarized in Table 3. Some backcross animals developed an early pronounced increase of UAE (maximum value observed 33.29 mg/24 h) at 8 wk of age. At 14 and 24 wk of age, some adult animals demonstrated values for UAE and UPE similar to those observed in parental MWF rats. The values for SBP ranged from 101 to 189 mmHg. The analysis demonstrated no significant correlation between SBP and UAE at 14 wk of age (r = 0.1, P = 0.15). The mean number of superficial glomeruli was 2.5 ± 1.9, and the mean number of surface glomeruli was 0.75 ± 1.0.


View this table:
[in this window]
[in a new window]

  		Table 3. Descriptive statistics for urinary albumin excretion (UAE), protein excretion (UPE), and renal histology parameters in backcross animals
 
The descriptive statistics for GSI and RIF in backcross animals are presented in Table 3. The range of GSI values were in keeping with the data observed in the parental strains. The variance observed for RIF phenotypes was larger compared with GSI, as expected from the parental data, but the maximal values observed in backcross animals were lower compared with MWF (Figure 3). SBP demonstrated no significant correlation with either GSI or RIF (P > 0.05, respectively). UAE at 24 wk of age demonstrated no significant correlation with GSI (r = -0.09, P = 0.17), whereas a significant albeit moderate correlation between UAE and RIF was observed (r = 0.21, P = 0.0034).



View larger version (23K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 3. Rat chromosome linkage maps and QTL localization for urinary albumin excretion (UAE), urinary protein excretion (UPE), and number of superficial glomeruli (SfG) and surface glomeruli (SG). The position of the suggestive linkage for renal interstitial fibrosis is indicated by the arrow. Thick solid bars represent the 1-LOD-interval, and thin solid bars represent the 2-Lod-interval for placement of the QTL (22). Only chromosomes are shown with significant linkage values (LOD > 3.3). For each chromosome a genetic distance of 10 centiMorgans (cM) is indicated.

 
QTL Mapping and Linkage Analysis
To perform a complete genome scan, more than 300 markers were tested to obtain a total number of 236 polymorphic markers distributed in a 10-cM distance.

Linkage to BP
Total genome screen analysis revealed no suggestive or significant linkages between SBP and any marker position in the genome (data not shown). The highest LOD value for SBP was observed at D7Rat7. At this locus, homozygosity for the MWF allele accounted for a 6-mmHg increase in SBP compared with heterozygous animals. Although, the significance at this locus (LOD = 1.7, P < 0.0057) was still below the threshold for suggestive linkage, the same region demonstrated suggestive linkage to UAE, thus pointing to a possible link to both SBP and UAE at this locus.

Linkage to UAE and UPE
We detected eight QTL on rat chromosomes (RNO) RNO2, RNO4, RNO6, RNO7, RNO8, RNO9, RNO15, and RNO X, demonstrating suggestive or significant linkage to UAE. The linkage results according to the marker locus demonstrating the highest LOD scores at each QTL are summarized in Table 4, and the placement on the chromosomes for the significant QTL on RNO1, RNO6, RNO8, and RNO9 are shown in Figure 3. In general, UPE demonstrated linkage to the same chromosomal fragments, but with weaker statistical significance. One exception was detected on RNO15, where an additional QTL demonstrated suggestive linkage to UPE but no linkage to UAE (Table 4). Taken together, the four QTL with significant linkage on RNO1, RNO6, RNO8, and RNO9 explained 50% of the total variance of UAE in this backcross population. The strongest effects were observed on RNO6 and RNO8; the two QTL accounted for 33.5% of the total variance of UAE. The QTL on RNO6 appears of special importance, because it demonstrated significant linkage (LOD 4.3) already in young animals at 8 wk of age (Table 4). Second, homozygosity for the MWF allele at this QTL demonstrated a strong phenotypic effect and accounted for a threefold elevation of UAE compared with heterozygous animals at 24 wk of age. Third, a similar although weaker statistical and phenotypic effect on early onset UAE at 8 wk of age was recently demonstrated in the same region in a backcross population between MWF and normotensive Lewis animals (Figure 4) (4).


View this table:
[in this window]
[in a new window]

  		Table 4. Linkage results for UAE and UPE in backcross animalsa
 


View larger version (26K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 4. QTL mapping for urinary albumin excretion on rat chromosome 6. Comparison of the current backcross population between SHR and MWF (thick curves) and the previous population (4) between Lewis and MWF (thin curves) at 8 wk (dotted curves), 14 wk (broken curves), and 24 wk of age (solid curves), respectively. Dotted line at LOD = 1.9 indicates the threshold for suggestive linkage, and solid line at LOD = 3.3 indicates the threshold for significant linkage.

 
Linkage to Renal Histology Phenotypes
The linkage results for renal histology phenotypes are presented in Table 5. Three QTL with suggestive linkage to superficial glomeruli were detected. The chromosomal position for the only significant locus linked to both superficial and surface glomeruli on RNO6 was detected in an interval overlapping with the UAE QTL on this chromosome. (Figure 3).


View this table:
[in this window]
[in a new window]

  		Table 5. Linkage results for superficial glomeruli, and renal interstitial fibrosis in backcross animalsa
 
Total genome mapping identified no suggestive or significant linkage to GSI. The highest LOD score (1.7) was detected on RNO4 at the marker D4Rat72 outside the suggestive UAE QTL on this chromosome. In contrast, suggestive linkage to RIF was identified on RNO6 (Table 5). Homozygosity for the MWF allele accounted for a 15.8% increase of RIF compared with heterozygous animals (P = 0.0009). The correlation analysis between UAE and RIF according to genotypes at the marker locus D6Mit8 of the QTL on RNO6 is shown in Figure 5. It occurred that animals with the heterozygous genotype showed a significant correlation (P = 0.023), whereas animals with the homozygous genotype carrying two MWF alleles and demonstrating increased RIF values showed no significant correlation between UAE and RIF (P = 0.35, Figure 5).



View larger version (19K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 5. Correlation analysis between urinary albumin excretion (UAE) represented on a log scale and renal interstitial fibrosis in backcross animals at 24 wk of age. The black squares indicate the backcross animals that are homozygous for the MWF allele, and the open circles represent the animals that are heterozygous at marker D6Mit8. The solid correlation line represents the homozygous and the broken line the heterozygous animals. Backcross animals that are homozygous at D6Mit8 demonstrated no significant correlation (r = 0.1, P = 0.35), while heterozygous animals showed a significant moderate correlation (r = 0.245, P = 0.023).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Major progress in our understanding of the glomerular filtration barrier has recently been achieved by the molecular genetic analysis of rare inherited forms of renal diseases (16). A case in point represents the characterization of the role of nephrin (i.e., NPHS1) in patients with congenital nephrotic syndrome of the Finnish type (17). Recessive inheritance of two defective NPHS1 alleles results in marked proteinuria in affected children and clearly demonstrates the non-redundant role of nephrin as a pivotal component of the glomerular slit diaphragm and its importance for maintenance of glomerular permselectivity (16–18). In contrast, our experimental QTL mapping analysis of increased UAE in the MWF rat reveals a polygenetic determination in which the contributing QTL appear to be redundant and background-dependent. Hence, in our previous study contrasting the MWF rat with the normotensive Lewis rat as a low UAE strain, we identified three QTL with significant linkage to UAE on RNO1, RNO12, and RNO17 (4). A fourth QTL with suggestive linkage (maximum LOD = 2.7; see Figure 4) was identified on RNO6. Averaged UAE levels in this previous study were rather low and the manifestation of an appreciable increase of UAE depended on the epistatic interaction between QTL, which is in agreement with studies in other rat models with spontaneous albuminuria (proteinuria) (10,19,20).

In the current experiment in which MWF rats were studied in a different genetic background by using the SHR model as a contrasting strain, the previously (in the backcross between MWF and Lewis animals) identified QTL on RNO1, RNO12, and RNO17 were not linked to UAE. This demonstrates the importance of genetic background effects in the polygenetic determination of UAE. The QTL on RNO6, in contrast, appeared already of interest in the backcross between MWF and Lewis, because it showed almost suggestive linkage to early-onset albuminuria in young animals at 8 wk of age (Figure 4) (4). The potential importance of this QTL for early onset albuminuria was clearly confirmed in the current study. Moreover, in contrast to the previous report the QTL on RNO6 showed a strong phenotypic effect with significant and increasing LOD values in adult animals. The other strong QTL on RNO8 identified here demonstrated no significant linkage in the previous backcross between MWF and Lewis and is thus dependent on the SHR background. Both QTL on RNO6 and RNO8 and the QTL on RNO1 and RNO9 show a potential co-localization with UAE or UPE QTL that have recently been identified in experimental crosses with Dahl SS rats (10,20). SS rats demonstrate an early onset of increased UAE that is independent from salt-loading, and the data obtained in the MWF and SS strains point to the possibility that the two models with genetic albuminuria share common disease loci for this phenotype. The homologous regions in the human genome for the UAE QTL RNO6 and RNO8 map to human chromosomes 14q24 and 15q, respectively (http://www.rgd.mcw.edu/VCMAP/). The discussion on the co-localization of potential candidate genes in such QTL intervals is always somewhat premature by virtue of the relatively large size of the chromosomal fragments that may contain many known candidate genes as well as many genes of unknown functional relevance for renal disease. Within the QTL on RNO6, we could, however, identify by comparative mapping analysis candidates such as {alpha}-actinin1 (Actn1), transforming growth factor–{beta}3 (Tgfb3), and arginase 2 (Arg2). These candidates could probably influence the development of glomeruli (Tgfb3) or the permeability of the glomerular filtration barrier by modulating podocyte function (Actn1) or glomerular nitric oxide metabolism (Arg2). The role of these candidates and of all other genes that are localized in the chromosomal fragment identified on RNO6 have to be analyzed by functional analyses in future studies and most importantly by positional analysis in congenic strains (21,22).

Male and female MWF animals exhibit a significant sexual dimorphism in UAE, UPE, and the development of progressive renal disease (2,11). Fassi et al. (2) have previously shown that both male and female MWF rats exhibit a similar reduction in the number of glomeruli per kidney due to an inborn nephron deficit compared with normal Wistar rats, whereas only MWF males are prone to develop progressive renal disease. We have recently shown that the lower UAE observed in MWF female animals can be increased by a high-NaCl diet and a concomitant increase in SBP due to their salt-sensitive hypertension (11). Whether or not a Y-chromosomal effect contributes to this sexual dimorphism in UAE has not been addressed in our study, because we generated no reciprocal crosses in which both parental strains contributed their Y chromosome to the backcross population. We performed the backcross only in one direction by using male parental MWF animals only, such as that all male backcross animals carried the Y-chromosome from the MWF strain. Therefore, we cannot exclude the possibility that a Y-chromosomal effect contributes to the sexual dimorphism of UAE in the MWF model. A genetic effect of the Y-chromosome on BP regulation has indeed been shown to contribute to the hypertension in spontaneously hypertensive rats (23).

An important additional goal of this report was to analyze the genetic relation between UAE and RIF. By using the SHR strain as a reference for MWF, it was possible to contrast the two hypertensive strains not only for low and high UAE levels but also for low and high RIF values. The quantification of RIF in parental strains revealed that the amount of RIF was 2.7-fold higher in SHR compared with MWF in the face of similar SBP. One might conclude that the contrasting RIF phenotype between SHR and MWF is related to the increased UAE observed in the MWF model, because increased albumin reabsorption in proximal tubular cells has been shown to trigger an interstitial inflammatory response that may eventually result in increased RIF (6,7). To test this potential link between UAE and RIF, we performed cosegregation and linkage analyses for both parameters in our backcross population between MWF and SHR. Overall, we found a moderate correlation between UAE and RIF in the total backcross population (r = 0.21), indicating that only 4.4% of the total variance of RIF can be attributed to variation of UAE. Interestingly, the UAE QTL on RNO6, which was linked to early onset albuminuria and demonstrated a pronounced effect on UAE, was also linked to the amount of RIF. Homozygosity for the MWF allele at this locus led to early-onset albuminuria in young animals and to higher UAE and RIF levels in adult animals. From a pathophysiologic point of view, the linkage of the QTL on RNO6 to both early-onset UAE and RIF appears reasonable, because RIF is a long-term consequence of increased glomerular albumin filtration. This could also explain the finding that animals with two MWF alleles at the QTL on RNO6 demonstrated no significant correlation between UAE and RIF values, because UAE levels in these animals are shifted to higher levels; this precluded the correlation that was seen in heterozygous animals (Figure 5). The influence of this locus on early-onset albuminuria on the other hand was sufficient to establish genetic linkage to RIF in adult animals. Our study demonstrates the power of genetic cosegregation and linkage analyses for the dissection of physiologic pathways involved in renal disease progression and establishes a genetic link between early-onset albuminuria and RIF at the QTL on RNO6. Notwithstanding, it should not be dismissed that the QTL on RNO6 was also linked to the increased numbers of superficial and surface glomeruli, which represent additional traits that are inherited in the MWF rat (24). We can therefore not exclude the possibility that a common molecular variant on RNO6 is responsible for the overlapping QTL for UAE, UPE, RIF, superficial and surface glomeruli, and thus for both the structural and functional phenotypes linked to this chromosomal region. Whether or not this holds true can be tested in future studies by transferring the chromosomal fragment spanning these QTL on RNO6 from the SHR strain into the contrasting MWF background by breeding congenic strains reference (21,22).

Interestingly, in our previous study (4) and in the experiment presented here, higher numbers for homozygous compared with heterozygous animals were particularly observed at SSLP markers that are linked to superficial glomeruli (Table 5), while the expected genotype ratio in a backcross study is 1:1. Systematic genotyping errors could be excluded by our described method for genotype determination and by consistent data after duplicate and independent analyses. Thus, a potential biologic explanation could be that homozygosity at these loci leads to a selection advantage during development. On the other hand, it is still possible that these observations are only chance findings.

It is currently unclear whether genetic variation at the nephrin locus (i.e., NPHS1) or at other loci such as podocin (i.e., NPHS2) that are responsible for monogenetic forms of nephrotic syndromes in humans confer genetic susceptibility for the manifestation and progression of proteinuria in more common chronic nephropathies. A recent report excluded linkage of these candidates as susceptibility loci for end-stage renal disease in diabetic nephropathy (25). Animal models such as the MWF rat represent valuable models to substitute the investigation of the polygenetic basis of common forms of chronic proteinuric nephropathies (1,4). Indeed, susceptibility loci identified in rodent models have been shown to be predictive for human genetic studies using the comparative genomic approach (26,27). Such susceptibility loci may provide target regions for subsequent studies using single nucleotide polymorphisms and linkage disequilibrium mapping in association studies in human patient populations (28–30). Interestingly, a major QTL termed Rf-1, which has been linked to UAE and UPE in the Fawn Hooded rat (19,31), has recently been implicated as a susceptibility locus for chronic renal disease or renal dysfunction in several independent human populations (25,32,33). We propose that particularly the further characterization of the QTL on RNO6 characterized here could lead to the identification of new targets for both the manifestation and progression of chronic proteinuric nephropathies.


   Acknowledgments
 
We are grateful to Heidelinde Müller, Gabriele Siebert, and Bettina Lack for excellent laboratory assistance. This work was supported by the DFG (KR-1152/2–2) and by the BMBF (Nationales Genomforschungsnetz: Herz-Kreislaufnetz Standort Berlin, 01GS 0106). L. K. was a recipient of an educational grant from the German Balkan Initiative supported by Schering AG, Berlin, Germany.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Macconi D, Ghilardi M, Bonassi ME, Mohamed EI, Abbate M, Colombi F, Remuzzi G, 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: 477–489, 2000[Abstract/Free Full Text]
  2. Fassi A, Sangalli F, Maffi R, Colombi F, Mohamed EI, Brenner BM, Remuzzi G, Remuzzi A: Progressive glomerular injury in the MWF rat is predicted by inborn nephron deficit. J Am Soc Nephrol 9: 1399–1406, 1998[Abstract]
  3. Iordache BE, Imberti O, Foglieni C, Remuzzi G, Bertani T, Remuzzi A: Effects of angiotensin-converting enzyme inhibition on glomerular capillary wall ultrastructure in MWF/Ztm rats. J Am Soc Nephrol 5: 1378–1384, 1994[Abstract]
  4. Schulz A, Litfin A, Kossmehl P, Kreutz R: Genetic dissection of increased urinary albumin excretion in the munich wistar fromter rat. J Am Soc Nephrol 13: 2706–2714, 2002[Abstract/Free Full Text]
  5. Remuzzi G, Ruggenenti P, Benigni A: Understanding the nature of renal disease progression. Kidney Int 51: 2–15, 1997[Medline]
  6. Remuzzi G: A unifying hypothesis for renal scarring linking protein trafficking to the different mediators of injury. Nephrol Dial Transplant 6 [15 Suppl]: 58–60, 2000
  7. Hsu SI, Couser WG: Chronic progression of tubulointerstitial damage in proteinuric renal disease is mediated by complement activation: A therapeutic role for complement inhibitors? J Am Soc Nephrol 4: S186–S191, 2003[CrossRef]
  8. de Heer E, Sijpkens YW, Verkade M, den Dulk M, Langers A, Schutrups J, Bruijn JA, van Es LA: Morphometry of interstitial fibrosis. Nephrol Dial Transplant 6 [15 Suppl]: 72–73, 2000
  9. Muller GA, Zeisberg M, Strutz F: The importance of tubulointerstitial damage in progressive renal disease. Nephrol Dial Transplant 6 [15 Suppl]: 76–77, 2000
  10. Poyan MA, Siegel AK, Kossmehl P, Schulz A, Plehm R, De Brujin JA, De Heer E, Kreutz R: Early onset albuminuria in Dahl rats is a polygenetic trait that is independent from salt-loading. Physiol Genomics 14: 209–216, 2003[Abstract/Free Full Text]
  11. Kreutz R, Kovacevic L, Schulz A, Rothermund L, Ketteler M, Paul M: Effect of high NaCl diet on spontaneous hypertension in a genetic rat model with reduced nephron number. J Hypertens 18: 777–782, 2000[CrossRef][Medline]
  12. Rothermund L, Luckert S, Kossmehl P, Paul M, Kreutz R: Renal endothelin ET(A)/ET(B) receptor imbalance differentiates salt-sensitive from salt-resistant spontaneous hypertension. Hypertension 37: 275–280, 2001[Abstract/Free Full Text]
  13. Kreutz R, Hübner N, James MR, Bihoreau MT, Gaugier D, Lathrop GM, Ganten D, Lindpaintner K: Dissection of a quantitative trait locus for genetic hypertension on rat chromosome 10. Proc Natl Acad Sci USA 92: 8778–8782, 1995[Abstract/Free Full Text]
  14. Lander ES, Green P, Abrahamson J, Barlow A, Daly MJ, Lincoln SE, Newburg L: MAPMAKER: An interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics 1: 174–181, 1987[CrossRef][Medline]
  15. Lander E, Kruglyak L: Genetic dissection of complex traits: guidelines for interpreting and reporting linkage results. Nat Genet 11: 241–247, 1995[CrossRef][Medline]
  16. Reiser J, von Gersdorff G, Simons M, Schwarz K, Faul C, Giardino L, Heider T, Loos M, Mundel P: Novel concepts in understanding and management of glomerular proteinuria. Nephrol Dial Transplant 17: 951–955, 2002[Free Full Text]
  17. Kestila M, Lenkkeri U, Mannikko M, Lamerdin J, McCready P, Putaala H, Ruotsalainen V, Morita T, Nissinen M, Herva R, Kashtan CE, Peltonen L, Holmberg C, Olsen A, Tryggvason K: Positionally cloned gene for a novel glomerular protein–nephrin–is mutated in congenital nephrotic syndrome. Mol Cell 1: 575–582, 1998[CrossRef][Medline]
  18. Pavenstadt H, Kriz W, Kretzler M: Cell biology of the glomerular podocyte. Physiol Rev 83: 253–307, 2003[Abstract/Free Full Text]
  19. Shiozawa M, Provoost AP, van Dokkum RP, Majewski RR, Jacob HJ: Evidence of gene-gene interactions in the genetic susceptibility to renal impairment after unilateral nephrectomy. J Am Soc Nephrol 11: 2068–2078, 2000[Abstract/Free Full Text]
  20. Garrett MR, Dene H, Rapp JP: Time-course genetic analysis of albuminuria in Dahl salt-sensitive rats on low-salt diet. J Am Soc Nephrol 14: 1175–1187, 2003[Abstract/Free Full Text]
  21. Kreutz R, Hubner N: Congenic rat strains are important tools for the genetic dissection of essential hypertension. Semin Nephrol 22: 135–147, 2002[CrossRef][Medline]
  22. Rapp JP: Genetic analysis of inherited hypertension in the rat. Physiol Rev 80: 135–172, 2000[Abstract/Free Full Text]
  23. Kreutz R, Stock P, Struk B, Lindpaintner K: The Y chromosome: Epistatic and ecogenetic interactions in genetic hypertension. Hypertension 28: 895–897, 1996[Abstract/Free Full Text]
  24. Hackbarth H, Gärtner K, Alt JM, Stolte H: A subline of the Munich Wistar (MW) strain: response to selection for surface glomeruli. Rat News Lett 7: 1378–1384, 1980
  25. Iyengar SK, Fox KA, Schachere M, Manzoor F, Slaughter ME, Covic AM, Orloff SM, Hayden PS, Olson JM, Schelling JR, Sedor JR: Linkage analysis of candidate loci for end-stage renal disease due to diabetic nephropathy. J Am Soc Nephrol 14: S195–S201, 2003[Abstract/Free Full Text]
  26. Stoll M, Kwitek-Black AE, Cowley AWJ, Harris EL, Harrap SB, Krieger JE, Printz MP, Provoost AP, Sassard J, Jacob HJ: New target regions for human hypertension via comparative genomics Genome Res 10: 473–482, 2000[Abstract/Free Full Text]
  27. Bihoreau MT, Megel N, Brown JH, Kranzlin B, Crombez L, Tychinskaya Y, Broxholme J, Kratz S, Bergmann V, Hoffman S, Gauguier D, Gretz N: Characterization of a major modifier locus for polycystic kidney disease (Modpkdr1) in the Han: SPRD(cy/+) rat in a region conserved with a mouse modifier locus for Alport syndrome. Hum Mol Genet 11: 2165–2173, 2002[Abstract/Free Full Text]
  28. Risch N, Merikangas K: The future of genetic studies of complex human diseases. Science 273: 1516–1517, 1996[Abstract/Free Full Text]
  29. Barton A, Eyre S, Myerscough A, Brintnell B, Ward D, Ollier WE, Lorentzen JC, Klareskog L, Silman A, John S, Worthington J: High resolution linkage and association mapping identifies a novel rheumatoid arthritis susceptibility locus homologous to one linked to two rat models of inflammatory arthritis. Hum Mol Genet 10: 1901–1906, 2001[Abstract/Free Full Text]
  30. Freedman BI: Susceptibility genes for hypertension and renal failure. J Am Soc Nephrol 14: S192–S194, 2003[Abstract/Free Full Text]
  31. Brown DM, Provoost AP, Daly MJ, Lander ES, Jacob HJ: Renal disease susceptibility and hypertension are under independent genetic control in the fawn-hooded rat. Nat Genet 12: 44–51, 1996[CrossRef][Medline]
  32. Hunt SC, Hasstedt SJ, Coon H, Camp NJ, Cawthon RM, Wu LL, Hopkins PN: Linkage of creatinine clearance to chromosome 10 in Utah pedigrees replicates a locus for end-stage renal disease in humans and renal failure in the fawn-hooded rat. Kidney Int 62: 1143–1148, 2002[CrossRef][Medline]
  33. Freedman BI, Rich SS, Yu H, Roh BH, Bowden DW: Linkage heterogeneity of end-stage renal disease on human chromosome 10. Kidney Int 62: 770–774, 2002[CrossRef][Medline]
Received for publication July 10, 2003. Accepted for publication September 21, 2003.




This article has been cited by other articles:


Home page
 Physiol. GenomicsHome page
D. H. T. IJpelaar, A. Schulz, J. Aben, A. van der Wal, J. A. Bruijn, R. Kreutz, and E. de Heer
Genetic predisposition for glomerulonephritis-induced glomerulosclerosis in rats is linked to chromosome 1
Physiol Genomics, October 7, 2008; 35(2): 173 - 181.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. GenomicsHome page
A. Schulz, J. Hansch, K. Kuhn, M. Schlesener, P. Kossmehl, J. R. Nyengaard, N. Wendt, M. Huber, and R. Kreutz
Nephron deficit is not required for progressive proteinuria development in the Munich Wistar Fromter rat
Physiol Genomics, September 17, 2008; 35(1): 30 - 35.
[Abstract] [Full Text] [PDF]

Home page
 GeneticsHome page
C. Doorenbos, S.-W. Tsaih, S. Sheehan, N. Ishimori, G. Navis, G. Churchill, K. DiPetrillo, and R. Korstanje
Quantitative Trait Loci for Urinary Albumin in Crosses Between C57BL/6J and A/J Inbred Mice in the Presence and Absence of Apoe
Genetics, May 1, 2008; 179(1): 693 - 699.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
D. H. T. IJpelaar, A. Schulz, K. Koop, M. Schlesener, J. A. Bruijn, D. Kerjaschki, R. Kreutz, and E. de Heer
Glomerular hypertrophy precedes albuminuria and segmental loss of podoplanin in podocytes in Munich-Wistar-Fromter rats
Am J Physiol Renal Physiol, April 1, 2008; 294(4): F758 - F767.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. GenomicsHome page
M. R. Garrett, W. T. Gunning, T. Radecki, and A. Richard
Dissection of a genetic locus influencing renal function in the rat and its concordance with kidney disease loci on human chromosome 1q21
Physiol Genomics, August 20, 2007; 30(3): 322 - 334.
[Abstract] [Full Text] [PDF]

Home page
 J. Am. Soc. Nephrol.Home page
A. Schulz, J. Weiss, M. Schlesener, J. Hansch, M. Wehland, N. Wendt, P. Kossmehl, A. Sietmann, D. Grimm, M. Stoll, et al.
Development of Overt Proteinuria in the Munich Wistar Fromter Rat Is Suppressed by Replacement of Chromosome 6 in a Consomic Rat Strain
J. Am. Soc. Nephrol., January 1, 2007; 18(1): 113 - 121.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. GenomicsHome page
M. R. Garrett, B. Joe, and S. Yerga-Woolwine
Genetic linkage of urinary albumin excretion in Dahl salt-sensitive rats: influence of dietary salt and confirmation using congenic strains.
Physiol Genomics, March 13, 2006; 25(1): 39 - 49.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. GenomicsHome page
C. Yagil, M. Sapojnikov, A. Wechsler, A. Korol, and Y. Yagil
Genetic dissection of proteinuria in the Sabra rat
Physiol Genomics, March 13, 2006; 25(1): 121 - 133.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
W. Yu, R. M. Sandoval, and B. A. Molitoris
Quantitative intravital microscopy using a Generalized Polarity concept for kidney studies
Am J Physiol Cell Physiol, November 1, 2005; 289(5): C1197 - C1208.
[Abstract] [Full Text] [PDF]

Home page
 J. Am. Soc. Nephrol.Home page
A. Rangel-Filho, M. Sharma, Y. H. Datta, C. Moreno, R. J. Roman, Y. Iwamoto, A. P. Provoost, J. Lazar, and H. J. Jacob
Rf-2 Gene Modulates Proteinuria and Albuminuria Independently of Changes in Glomerular Permeability in the Fawn-Hooded Hypertensive Rat
J. Am. Soc. Nephrol., April 1, 2005; 16(4): 852 - 856.
[Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
R. Korstanje and K. DiPetrillo
Unraveling the genetics of chronic kidney disease using animal models
Am J Physiol Renal Physiol, September 1, 2004; 287(3): F347 - F352.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Schulz, A.
Right arrow Articles by Kreutz, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schulz, A.
Right arrow Articles by Kreutz, R.

HOME CURRENT ISSUE ARCHIVES JASN Express ONLINE SUBMISSION AUTHOR INFO
EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP