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Physiol. Genomics 25: 39-49, 2006; doi:10.1152/physiolgenomics.00150.2005
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Received 30 June 2005; accepted in final form 1 January 2006.
Physiological Genomics 25:39-49 (2006)
American Physiological Society © 2006 American Physiological Society

Genetic linkage of urinary albumin excretion in Dahl salt-sensitive rats: influence of dietary salt and confirmation using congenic strains

Michael R. Garrett, Bina Joe and Shane Yerga-Woolwine

Department of Physiology and Cardiovascular Genomics, Medical University of Ohio, Toledo, Ohio


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Previously, we reported a linkage analysis for urinary albumin excretion (UAE) from a backcross population derived from the Dahl salt-sensitive (S) rat and the spontaneously hypertensive rat (SHR) raised on a low-salt diet. The present study sought to examine the effect of salt loading on the observation of UAE quantitative trait loci (QTL) using a F1(S x SHR) x S backcross population (n = 228) raised on a 2% NaCl diet. Parental strain data demonstrated that S rats have significantly higher blood pressure (BP) and UAE compared with either F1(S x SHR) or SHR at 8 wk of age, and this difference was exacerbated by 12 wk of age in response to a high-salt diet (2% NaCl). Genome scans done at 8, 12, and 16 wk of age yielded eight QTL for UAE. At week 8 (low salt), QTL for UAE were observed on rat chromosomes (RNO) 1, 2, 6, 8, 9, 11, 13, and 19. Week 8 linkage analysis confirmed previous linkage data and provided a baseline to examine the effect of salt loading at subsequent time points. At weeks 12 and 16 (after salt- loading), QTL for UAE were observed on RNO1, -6, -8, -9, and -13. Surprisingly, UAE QTL were no longer observed on RNO2, -11, and -19 after salt loading, suggesting that these QTL are attenuated by increased salt intake. The effects of UAE QTL on RNO2, -6, -9, -11, and -13 were examined using congenic strains whereby the SHR alleles at each QTL were placed on the S background. These congenic strains demonstrated large and significant effects on UAE compared with the S rat, proving that QTL for UAE reside on these chromosomes.

spontaneously hypertensive rats; albuminuria; proteinuria; blood pressure; hypertension; quantitative trait loci; kidney weight


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
SALT-SENSITIVE HYPERTENSION is known to be a contributing factor for the progression of kidney disease as well as for cardiovascular diseases (5, 15, 21). Clinical studies (2, 6, 8, 22) have suggested that those patients who exhibit salt-sensitive hypertension are more prone to develop severe hypertension-related target organ damage. This observation is supported by experimental rat models that exhibit salt-sensitive hypertension and associated renal damage. One such model is the Dahl salt-sensitive (S) rat. The S rat was originally bred for sensitivity to the hypertensive effects of a high-salt diet (8% NaCl) (7). In response to a high-salt diet, these animals exhibit a marked and rapid increase in blood pressure (BP) associated with severe renal damage (28, 34, 35). On a low-salt diet, these rats still develop hypertension and renal damage with age (12), but the onset and progession is slower compared with their response on a high-salt diet. The spontaneous hypertensive rat (SHR) offers an interesting contrast to the S rat because despite developing hypertension, it is resistant to the development of renal damage (11, 12).

It is well known that an early sign of deteriorating kidney function in certain disease states is the presence of small amounts of albumin in the urine (24). As kidney function declines, the amount of albumin in the urine increases and eventually develops into the abnormal range, referred to as "albuminuria." This threshold is well defined in humans (24), but it is not so clear for studies involving rodents. For the purpose of our work, urinary albumin excretion (UAE) was used as a marker of kidney function without necessarily implying the existence of a disease state.

We previously conducted a linkage analysis for UAE using a backcross population derived from S and SHR raised on a low-salt diet (12). The study identified eight quantitative trait loci (QTL) where the S rat allele increased UAE and two QTL where the SHR allele increased UAE.

The aim of the present study was to perform a second linkage analysis to determine if QTL for UAE would be influenced by salt loading either by altering the time-course pattern of known QTL or by identifying QTL that were not detected with low salt. A second aim was to perform congenic strain analysis for several UAE QTL to confirm the linkage analysis [on rat chromosomes (RNO)2, -6, -9, -11, and -13] and to demonstrate the magnitude of the effect of each QTL once it was isolated on the S background.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals
All procedures were carried out with approval from our Institutional Animal Care and Use Committee. Inbred Dahl salt-sensitive (SS/Jr or S) rats were maintained in our animal facility at the Medical University of Ohio (Toledo, OH). Spontaneously hypertensive rats (SHR/NHsd or SHR) were originally obtained from Harlan Sprague Dawley (Indianapolis, IN) and have been inbred for >20 generations at the Medical University of Ohio. To determine the effect of salt loading in F1(S x SHR) animals on systolic BP, urinary protein excretion (UPE), and UAE, S females were crossed to SHR males to produce F1(S x SHR) animals. Two groups of age-matched S, F1, and SHR male rats (n = 6 rats for each strain per group) were studied for BP, UPE, and UAE at 12 wk of age. Group 1 was fed a low-salt diet (0.3% NaCl, TD 7034, Harlan Teklad; Madison, WI) from weaning (30 days), and group 2 animals were on a low-salt diet from weaning until 8 wk of age and then fed a 2% NaCl diet (TD 94217, Harlan Teklad) for 4 wk.

A F1(S x SHR) x S backcross population of 228 male rats was produced by crossing F1 females with S males. The backcross animals were weaned at 30 days of age and placed on a low-salt diet (0.3% NaCl) until the animals were 8 wk of age. At 8 wk of age, animals were studied for BP, UPE, and UAE. Once testing was complete, animals were placed on a high-salt diet (2% NaCl) for the duration of the experiment. The animals were studied again for BP, UPE, and UAE at 12 and 16 wk of age.

The 228 rats were handled in 4 blocks of 57 animals. Rats in each block were aged matched, and there was little difference in age between blocks. This allowed for phenotyping on all rats at approximately the same ages. Block effects, if any, were removed statistically.

Rats were killed by CO2 inhalation. Livers were harvested and archived at –80°C for subsequent DNA extraction. Body weight (BW), heart weight (HW), and total kidney weight (TKW) were measured. The right kidney was fixed in 10% buffered formalin and embedded in paraffin for sectioning and staining.

Congenic Strains
Congenic strains were developed using a speed-congenic approach (19, 37), whereby a genome scan using 100–114 microsatellite markers was performed at each generation. This approach allowed for the selection of animals that had incorporated the greatest amount of background genome (S alleles) while still containing the introgressed region of interest (alleles from SHR). Female S rats were bred with male SHR to produce F1 rats. Female F1 rats were backcrossed to male S rats (ensuring that all subsequent male animals had a Y chromosome derived from the S rat) to produce the first backcross generation (BC1). This is important in dealing with SHR because the SHR Y chromosome has been shown to influence BP in some experimental crosses (10) and differences in BP between strains could confound the analysis of renal damage.

At BC1, animals were selected that retained the greatest amount of S genome while being heterozygous S/SHR for either RNO2, -6, -9, -11, or -13. These animals were designated as the best breeders for subsequent pairing with S rats to produce BC2 animals. The same process continued for BC2 through BC5. At BC5, the background genome for each strain that had the S/SHR genotype on either RNO2, -6, -9, -11, or -13 was found to be homozygous SS. These animals were bred to S rats to generate additional animals heterozygous for the introgressed region. The heterozygous animals for each region (either RNO2, -6, -9, -11, or -13) were intercrossed, and animals that were homozygous for SHR alleles throughout the introgressed region were selected to fix the SHR alleles on the S background. The five strains produced are denoted as S.SHR(2), S.SHR(6), S.SHR(9), S.SHR(11), and S.SHR(13).

For testing, S, SHR, and all five congenic strains were bred concomitantly. At 30 days of age, rats were weaned and placed on a low-salt diet. Urine was collected on both male and female animals at 51–53 days of age for determination of UPE and UAE.

Phenotyping
Blood pressure.
BP was measured by the tail-cuff method on conscious restrained rats warmed to 28°C. The procedure for BP determination for the initial study involving the effects on salt loading on S, F1 and SHR rats and for the backcross population was the same. Of the 57 rats in each block for the backcross population (as noted above), a subset of 48 animals was selected at random from each block for BP determination. A total of 192 animals had their BP measured. Starting at 8 wk of age, BP was measured on 2 consecutive days by two operators. Each operator had one session with each rat. The final BP of each rat was the average of both sessions. The procedure was repeated at weeks 12 and 16.

UPE and UAE determination.
To collect urine, animals were kept in metabolism cages (Lab Products; Seaford, DE) for 24 h with free access to water. Sodium azide was added to the collection vials for a final concentration of ~0.01% in the urine. Total UPE was determined colorimetrically using a pyrogallol red-molybdate complex (Quantimetrix; Redondo Beach, CA) and expressed as milligrams of protein per 24 h. UAE was determined by a rat albumin EIA kit (SPI-bio) and also by SDS-PAGE, as reported previously (12). UAE is expressed as milligrams of albumin per 24 h.

Histology.
Kidneys were cut into 3-µm sections and stained with hematoxylin and eosin. One central longitudinal section from the right kidney was examined in a blinded fashion on an arbitrary semiquantitative scale from 0 to 4 for kidney lesions as reported earlier (12). The grades represent a visually integrated assessment of the severity and extent of the lesions, and no attempt was made to grade the individual components (e.g., glomeruli, tubules, and vasculature) separately. The primary use of the histology data was to serve as a confirmatory phenotype and as an indication of whether or not QTL for UPE were associated with any overt histological changes. The kidney lesion grades (KLG) were 0 (normal), 1 (mild), 2 (moderate), 3 (marked), and 4 (severe). It was possible to assign half grades so the grades were 0, 0.5, 1, 1.5, etc.

Genotyping
Genomic DNA for the backcross population (liver) or for the development of congenic strains (tail biopsy) was prepared using DNeasy 96 tissue kits (Qiagen; Valencia, CA). Genotyping was done using two methods: 1) a standard approach using agarose or polyacrylamide gel electrophoresis as previously described (9); and 2) a fluorescent-based approach using a Beckman Coulter CEQ8000XL capillary sequencer (3). In the the fluorescent-based approach, the forward primer of a microsatellite marker is tagged 5' with a M13 primer (5'-CACGACGTTGTAAAACGAC-3'). The M13 forward primer is used in combination with a fluorescently labeled M13 primer (dye D4-PA, Beckman Coulter; Fullerton, CA) to produce a PCR product that can be detected using the CEQ system (3). The M13 dye-labeled primer can be used with any PCR primer that is 5' tagged with M13, eliminating the need to purchase individually labeled primer sets.

PCRs for the fluorescent-based approach were prepared as follows in a 10-µl reaction: 1x buffer containing 1.5 mM MgCl2 (Promega; Madison, WI), 0.2 mM dNTPs (Sigma; St. Lois, MO), 2.5 pmol M13-D4 labeled dye (Research Genetics; Huntsville, AL), 0.1 pmol M13 forward primer, 2.5 pmol reverse primer, and 0.25 units Taq DNA polymerase (Promega). PCR amplification was performed as follows: 95°C for 5 min and continued for 5 cycles of 94°C for 40 s, 55°C for 40 s, and 72°C for 1 min, after which an additional 30 cycles of 94°C for 40 s, 50°C for 40 s, and 72°C for 1 min was performed. After amplification, the PCR products were diluted 1:10 in water, and 1 µl of each diluted product (4–6 different PCR products ranging in size from 100 to 400 bp) was added to 40 µl of sample loading solution (SLS) buffer (Beckman Coulter) containing a DNA size standard, loaded onto a CEQ sequencer, and then analyzed using CEQ fragmentation analysis software.

A complete genome scan of all 21 chromosomes (except the Y chromosome) was performed using a total of 174 markers that were approximately evenly spaced throughout the genome. The information on microsatellites markers were obtained from the following databases: 1) the Rat Genome Database at the Medical College of Wisconsin (http://www.rgd.mcw.edu); 2) the Wellcome Trust Centre for Human Genomics (Oxford, UK) (http://www.well.ox.ac.uk); and 3) the Department of Physiology and Cardiovascular Genomics at the Medical University of Ohio (http://www.meduohio.edu/depts/physiology/research).

Linkage and Statistical Analysis
Linkage analysis and QTL localization were performed using Map Manager QTX (http://mapmgr.roswellparks.org) (18). The likelihood ratio statistic (LRS) generated by Map Manager QTX as a measure of the significance of a QTL was converted into a LOD score (LOD = LRS/4.6) for reporting purposes. For a backcross population, a LOD score of at least 1.9 was considered evidence for suggestive linkage and a LOD score of 3.3 or above indicated significant linkage between phenotype and genotype (17). Determination of the "phenotypic effect" (average effect of S/S animals minus average effect of S/SHR animals) for phenotypes with suggestive or significant linkage to a chromosome was done by selecting one index marker at or near the QTL peak and performing an independent t-test using SPSS (Chicago, IL). KLG grade scores were additionally analyzed by a nonparametric Kruskal-Wallis test.

All other data were analyzed by one-way ANOVA followed by post hoc multiple comparisons using the Tukey's test (SPSS). All data are presented as means ± SE.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of a High-Salt Diet on BP, UPE, and UAE in S, F1(S x SHR), and SHR
Data for BP, UPE, and UAE for both parental strains and F1(S x SHR) are presented in Fig. 1. On a low-salt diet, S and SHR at 12 wk of age showed similar BP values (Fig. 1A). F1 rats had BP values slightly lower than either of the parental strains. On a high-salt diet, hypertension in the S rat was exacerbated, but no significant effect was seen for F1 or SHR. The BP of 12-wk-old salt-loaded S rats was 38 ± 10.1 mmHg (P < 0.001), higher than low-salt S rats of the same age (Fig. 1A).


Figure 1
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  		Fig. 1. Effect of a high-salt (HS) diet (2% NaCl) on systolic blood pressure (BP; A), urinary protein excretion (UPE; B), and urinary albumin excretion (UAE; C) for Dahl salt-sensitive (S), F1(S x SHR), and spontaneously hypertensive rats (SHR) at 12 wk of age. Salt-loaded rats were placed on a HS diet (2% NaCl) at 8 wk of age and continued through 12 wk of age. The low-salt (LS) and HS groups were raised and studied concomitantly. In B and C, the dashed lines represent the thresholds for "proteinuria" (>20 mg/24 h) and "albuminuria" (>5 mg/24 h), respectively. Statistical analysis was done using one-way ANOVA followed by post hoc multiple comparisons using the Tukey's test. Values are means ± SE; n = 6 rats/group. aSignificantly different from each other; bnot significantly different from each other; csignificantly different from b (P < 0.01).

 
In contrast to BP, UPE and UAE levels showed a significant difference between strains even on a low-salt diet (Fig. 1, B and C). For the purposes of our analysis, we defined UPE >20 mg/24 h as "proteinuria" and UAE > 5 mg/24 h as "albuminuria." The proteinuria threshold has been used previously by others (38), and values below either of these thresholds appear to correspond to the normal range of UPE and UAE in several rat inbred strains (25). These threshold values provide some value with regard to classifying what is normal and what is abnormal. S rat UPE was 109 ± 10.5 mg/24 h, clearly well above the threshold to be considered as proteinuria. The S rat had fivefold higher UPE than either the F1 hybrid (17 ± 2.1 mg/24 h) or SHR (19 ± 1.50 mg/24 h) (Fig. 1B). Remarkably, F1 UPE was below the 20 mg/24 h threshold and would be considered normal. The UAE difference between the strains was dramatically larger than UPE. However, UAE for the F1 or SHR are below what would be considered albuminuria and fall in the normal range. There was no significant difference between F1 and SHR for either UPE or UAE on a low-salt diet.

On a high-salt diet, UPE in the S rat was greatly influenced by salt loading. The UPE for 12-wk-old salt-loaded S rats was 53 ± 10.1 mg/24 h (P < 0.0001) higher than low-salt S rats of the same age. Neither F1 nor SHR animals experienced a significant increase in UPE beyond their low-salt values. Consequently, there was a greater fold difference between the S rat and F1 or SHR compared with low-salt feeding. The S rat had approximately sevenfold higher UPE than either the F1 hybrid (19.5 ± 1.1 mg/24 h) or SHR (22.6 ± 3.2 mg/24 h) (Fig. 1B). Similarly, UAE increased significantly in the S rat (Fig. 1C) but remained unchanged in the F1 or SHR.

Effect of a High-Salt Diet on BP and UAE on the F1(S x SHR) x S Population
A backcross population (n = 228) of male rats was challenged with a high-salt diet (2% NaCl) to study the effect of BP changes on kidney function, i.e., UPE and UAE. The population was initially raised on a low-salt diet until week 8 phenotyping was complete, and the animals were then placed on a 2% NaCl diet until week 16.

Figure 2 illustrates the effect that a high-salt diet had on the backcross population for BP and UAE compared with the previously published low-salt population (12). For the low-salt population, there was a slight but significant increase in BP from 146 ± 1.1 mmHg in week 8 to 155 ± 1.2 mmHg in week 12 (Fig. 2A). From week 8 to 12, there was also a slight increase in the proportion of animals with higher BP. In contrast, the BP of the high-salt population (after 4 wk of salt loading) markedly increased from 140 ± 1.1 to 165 ± 1.5 mmHg, a difference of 24.7 ± 1.9 mmHg (P < 0.0001). Additionally, there was a dramatic shift in the number of animals that had higher BP compared with animals in week 8 (Fig. 2A).


Figure 2
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  		Fig. 2. Box plot comparing BP and UAE between the backcross population raised on a LS diet [previously published (12)] and the backcross population raised on a HS diet (this study). For the HS population, rats were maintained on a LS diet until 8 wk of age and then placed on a 2% NaCl diet until 16 wk of age. Open plots show week 8 data and shaded plots show week 12 data. The box itself contains the middle 50% of the data. The upper hinge indicates the 75th percentile of the data set and the lower hinge represents the 25th percentile. The line in the box indicates the median value of the data. The + symbol represents the mean value of the population.

 
At week 8, the average UAE value for the low-salt population was 3.6 ± 0.32 mg/24 h, and this essentially remained unchanged (3.9 ± 0.35 mg/24 h) through week 12 (Fig. 2B), even though average BP in the population was significantly higher than at week 8. In comparison, average UAE for the high-salt population (after 4 wk of salt loading) tripled from 3.4 ± 0.32 to 9.6 ± 0.66 mg/24 h, presumably as a result of the large increase in average BP in the population.

Figure 3 gives a more detailed presentation of the characteristics of the present backcross population including data for BP, UPE, UAE, BW, HW, TKW, and KLG at each time point studied. The main point to note in Fig. 3 is that many rats had UPE and UAE values that were above the normal range of ~20 mg/24 h for UPE and above ~5 mg/24 h for UAE. Thus a majority of animals in this population would be classified as having proteinuria and albuminuria based on these thresholds. Additionally, the scatterplots for UPE and UAE show that the distributions for each measure are skewed to the right (i.e., more lower values). For this reason, subsequent analysis was carried out on the natural logarithm (ln) of UPE and UAE data to approximate a normal distribution.


Figure 3
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  		Fig. 3. Scatterplots showing time-course population data for systolic BP, UPE, UAE, and body weight (BW) for the backcross population raised on a HS diet. For this HS population, rats were maintained on a LS diet until 8 wk of age and then placed on a 2% NaCl diet until 16 wk of age. At week 16, backcross animals were killed, and heart weight (HW), total kidney weight (TKW), and kidney lesion grade (KLG) were measured. HW, TKW, and KLG data are depicted in the histograms. The arrowheads represent the location of the mean value of the population. Values are means ± SD.

 
Time-Course Genome Scan for BP, UPE, and UAE on the F1(S x SHR) x S Population Raised on a High-Salt Diet
Genome scans for BP, UPE, and UAE were done at 8, 12, and 16 wk of age for the backcross population maintained on a high-salt diet (2% NaCl). Additionally, HW, TKW, and KLG were studied at week 16 after the animals were killed. Figure 4 gives a summary of the QTL identified at all time points. Suggestive or significant QTL with linkage to UPE (gray line, Fig. 4) were identified on RNO2, -6, and -9 for week 8. UAE (red line, Fig. 4) QTL colocalized with UPE for these chromosomes and were also observed on RNO1, -8, -11, -13, and -19. At week 12, after 4 wk of a high-salt diet, QTL for UPE and UAE were observed on RNO1, -6, -8, and -9. QTL on RNO13 was only observed for UAE. QTL for UPE and UAE on RNO2, -11, and -19 were no longer observed after 4 wk of a high-salt diet. The same UPE and UAE QTL observed at week 12 were also seen at week 16 (8 wk of a high-salt diet). Only one suggestive QTL for BP was observed on RNO11 at week 12 but not at week 16.


Figure 4
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  		Fig. 4. Summary of genome scan for systolic BP, UPE, UAE, KLG, HW, and TKW at 8, 12 and 16 wk of age for the backcross population (n = 228) raised on a 2% NaCl diet. Animals were placed on the 2% NaCl diet after the initial phenotype data were collected for BP, UPE, and UAE at 8 wk of age. Thus only the data at the time points of 12 and 16 wk were from rats on the HS diet. The lines to the right of the linkage maps represent the 2-LOD interval of each quantitative trail loci (QTL) identified that achieved at least suggestive significance (LOD >1.9). *QTL that achieved a LOD >3.3. Traits that were observed at only one or two time points are labeled with the time points at which the QTL was observed. If no time point is specified, then the QTL was observed at all three time points.

 
At 16 wk of age, backcross animals were killed, and their kidneys were scored for KLG. The KLG represents an integrated assessment of kidney pathology and was intended solely as a confirmatory phenotype to UPE and UAE. Most UPE and UAE were associated with a QTL for KLG, demonstrating that it at least served this limited purpose. KLG (purple line, Fig. 4) QTL were observed on RNO1, -2, -8, -9, and -13. The KLG QTL on RNO8 was the only KLG QTL to meet the criteria for significant linkage (LOD > 3.3). All other KLG QTL reached only a suggestive level of significance. Overall, the UPE effects at each QTL were modest, in the range of 2–12 mg/24 h, depending on the time point (Table 1). The UAE effects were in the range of 1–3 mg/24 h. The effect of each UPE or UAE QTL at week 12 was approximately threefold higher than that of week 8 (Table 1). Additionally, the phenotypic effect at each QTL was slightly higher for week 16 compared with week 12 for UPE but remained unchanged for UAE. The UAE QTL on RNO8 had the largest effect of all the QTL observed and ranged from 8 to 12 mg/24 h (weeks 12–16). The RNO8 QTL accounted for 8% of the phenotypic variance in UAE (out of ~40% from all QTL).


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  		Table 1. Phenotypic effect by genotype for BP, UPE, UAE, HW, TKW, and KLG in the backcross population of 228 rats raised on a 2% NaCl diet

 
The S/S genotype was associated with increased UPE and UAE at QTL detected on RNO1, -2, -6, -8, -9, -13, and -19 (Table 1). However, for the QTL on RNO11, the S/SHR genotype was associated with increased UPE and UAE (i.e., the SHR allele was associated with increased UPE/UAE and the S allele was associated with decreased UPE/UAE). The KLG effect at each QTL was modest (~0.5 units). The direction of the effect of each KLG QTL was always concordant with that of the UPE and UAE QTL (Table 1).

Phenotypic Analysis of Congenic Strains
UPE and UAE data for S, SHR, and five congenic strains are shown in Fig. 5 and correspond to the QTL analysis performed at week 8 before the animals were placed on a high-salt diet. Both male and female rats were tested to examine the effect of sex on each QTL. Although data are only shown for male rats, the results between males and females were consistent. The S.SHR(2) congenic strain had significantly lower (P < 0.0001) UPE and UAE compared with the S strain (Fig. 5A). UPE for the S.SHR(2) congenic strain was not significantly different from the SHR strain at this age. However, UAE was significantly different between the congenic and SHR strains. The S.SHR(2) congenic strain lowered UPE and UAE by ~75% compared with control S rats. This is the direction of change expected based on the genome scan (i.e., the SHR allele significantly lowered UPE compared with the S allele on RNO2). The RNO2 congenic strain had the largest effect in reducing UPE compared with the other four congenic strains.


Figure 5
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  		Fig. 5. Congenic (C) strain data for UPE and UAE QTL on rat chromosomes (RNO)2 (A), -6 (B), -9 (C), -11 (D), and -13 (E). The introgressed region of each congenic strain is shown next to the linkage map. The solid bars designate the extent of introgressed SHR alleles on the S background. The open regions on the ends of these bars represent the interval in which recombination occurred. Bar graphs show the effect of each congenic strain compared with both parental strains: S and SHR. The left y-axis plots the effect of UPE, and the right y-axis plots the effect of UAE. All the rats studied were bred concomitantly and age matched for urine collection. At 30 days of age, rats were weaned and placed on a LS (0.3% NaCl) diet. Urine was collected from animals 51–53 days of age for determination of UPE and UAE. The number of animals used for each strain were as follows: S, n = 18; S.SHR(2), n = 18; S.SHR(6), n = 16; S.SHR(9), n = 18; S.SHR(11), n = 12; and S.SHR(13), n = 16. Values are means ± SE. *S vs. C comparison, significantly different at P < 0.0001; #C vs. SHR comparison, significantly different at P < 0.0001. P values are from one-way ANOVA followed by post hoc multiple comparisons using the Tukey's test.

 
The congenic strains S.SHR(6), S.SHR(9), and S.SHR(13) had a similar reduction in UPE, ranging from 16 to 22 mg/24 h (Fig. 5, B, C, and E). However, all three strains had a greater fold reduction in UAE than UPE compared with control S rats. For example, the S.SHR(6) congenic strain had a 6.8-fold reduction in UAE compared with control S rats, whereas only a twofold difference was seen when UPE was compared between the two strains.

The S.SHR(11) congenic strain had significantly higher (P < 0.0001) UPE and UAE (Fig. 5D) compared with the control S strain. This QTL corresponds to the only QTL observed in the present study where the SHR allele was found to increase UPE and UAE. The S.SHR(11) congenic strain had a 65% increase in UPE over the control S strain. A 200% increase was observed when UAE was examined (Fig. 5D). Interestingly, this congenic strain was more susceptible to kidney damage (as measured by UPE and UAE) than the control S rats, which are already highly prone to kidney damage.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We have previously reported a time-course genome scan on a F1(S x SHR) x S population raised on a low-salt diet (12). The objective for the earlier study was to monitor the onset and development of UAE QTL while minimizing the effect of increased BP on renal damage. The present study sought to determine the effect of salt-induced high BP on the identification of UAE QTL. The backcross population, once placed on a 2% NaCl diet, did have a greater increase in average BP from week 8 to 12 than was observed in the low-salt population for the same period. The shift in the population's average BP from week 8 to 12 was modest but was associated with a large and significant effect on UAE. Interestingly, the population mean UAE was low (~10.0 mg/24 h) compared with the UAE of S rats at the same age (~80 mg/24 h), demonstrating that SHR alleles have a remarkable ability to confer resistance to UAE in the presence of the highly permissive S background, which readily develops hypertension and renal damage.

Figure 6 summarizes UAE QTL identified in the present study compared with QTL observed in the previously described low-salt population (12). For clarity, only UAE is presented, because all UPE QTL were concordant with UAE QTL. The initial time point (week 8 without salt loading) served to confirm the previous linkage analysis and also to provide a baseline for the effect of salt loading on the identification of UAE QTL.


Figure 6
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  		Fig. 6. Comparison of UAE QTL observed in the LS backcross population (12) and those observed in the HS backcross population (present study). The black boxes indicate that the QTL was observed at a given time point. The grey boxes indicate that the QTL was not observed.

 
The majority (8 of 9) of QTL for UAE found in the low-salt population were also observed in the present study, with the exception of the RNO10 UAE QTL. The RNO10 QTL was only detected at the suggestive level (LOD = 1.9–3.0) in the low-salt population at weeks 8 and 12, and the reduced number of animals in the high-salt population most likely lacked the statistical power to provide evidence for this QTL.

The UAE QTL identified after salt loading at weeks 12 and 16 were consistent with those found using the low-salt population (Fig. 6). UAE QTL on RNO1, -6, -8, -9, and -13 were seen throughout the period of salt loading, but those on RNO2, -11, and -19 were undetected in the high-salt population, demonstrating that these QTL were influenced by increased salt intake. For UAE QTL on RNO2 and -19, it is not readily apparent how salt loading acts to attenuate these QTL. However, a reasonable speculation can be made regarding the UAE QTL on RNO11. The SHR allele on RNO11 was found to be associated with increased UAE, and, once dietary NaCl was increased, the QTL was no longer observed, suggesting that the SHR allele on RNO11 no longer conferred susceptibly to UAE. It is known that the SHR has the ability to resist the development of proteinuria in the presence of systemic hypertension because it experiences increased renal vascular resistance largely due to preglomerular vasoconstriction (1, 14, 36). SHR alleles of RNO11 gene(s) may be a mediator of preglomerular vasoconstriction and protect the SHR from renal damage in response to an increased BP.

Congenic strain data confirmed the presence of UPE and UAE QTL on RNO2, -6, -9, -11, and -13 (comparable with week 8 of the genome scans). All the congenic strains had significantly different UPE and UAE compared with the S strain, with effects ranging from 15 to 40 mg/24 h for UAE. In contrast, for either genome scan, the effect of each QTL was at most 3.3 mg/24 h. This difference is best explained by the strong ability of the SHR alleles to exert their effects on the highly permissive S background.

The genetics of kidney disease have been studied using several rat models, and this work has been recently reviewed (16). Genetic studies involving the Buffalo (20, 23), Fawn hood hypertensive (FHH) (4, 31), Munich Wistar Frömter (29, 30), and Milan normotensive strain (MNS) (33) have consistently shown that high UAE is strongly recessive. This also holds true for the present work with the S rat. Many of these studies have not gone beyond the stage of linkage analysis, except for one study (27). One of the earliest linkage analysis came from a cross involving the FHH strain, in which five QTL for UAE were identified (Rf-1 through Rf-5) (4, 31). Interesting, Rangel-Filho et al. (27) recently published data suggesting that a naturally occurring mutation in the start codon of Rab32 (which leads to a knockout in the FHH strain) underlies the Rf-2 locus located on RNO1 (27). They suggested that Rab32 is involved in altering tubular reuptake and processing of filtered protein, although further studies are required to elucidate the causative mechanism.

Of particular interest are two linkage analyses that involved genome scans for UAE using F2(S x SHR) populations. The first F2(S x SHR) population was raised on a low-salt diet, and a genome scan identified UAE QTL on RNO2, -6, -8, -9, -10, -11, and -19 (26), consistent with the QTL we identified using a backcross population (12). A second F2(S x SHR) population was raised on a high-salt (4% NaCl) diet, and a genome scan identified UAE QTL on RNO3, -6, -8, -9, and -19 (32). Thus QTL for UAE on RNO2, -10, and -11 were not detected in the salt-loaded population but were observed in the low-salt F2 population. The present study confirms the observation that UAE QTL on RNO2 and -11 are influenced by salt loading. In contrast, Siegal et al. (32) detected the UAE QTL on RNO19 in both populations, whereas we no longer detected this QTL after salt loading.

Siegal et al. (32) found that QTL for BP colocalized with UAE on RNO3, -6, and -9. In contrast, no BP QTL were observed on either RNO6 or -9 in the present study but were observed on RNO11. BP QTL have also been observed on RNO3, -8, and -9 from a linkage analysis using a F2(S x SHR) population raised on a 8% NaCl diet (13). The BP QTL on RNO3 and -9 are consistent with those in a F2(S x SHR) population raised on 4% NaCl diet (32), but RNO6 is unique to the 4% NaCl population and RNO8 is unique to the 8% NaCl population. The discordance between the BP QTL observed between these studies is most likely a result of differences in the background genome (backcross vs. F2) and/or the degree of salt feeding (0.3%, 2%, 4%, or 8%). Taken together, data from all the crosses involving S and SHR suggest that UAE and BP QTL map to the same region on RNO6 and -8 and RNO9–11. The UAE QTL on RNO1, -2, -13, and -19 appear not to colocalize with BP QTL.

The UAE QTL reported in the present study control the trait of UAE, which is variable from low in the SHR to high in the S rat. From the data presented, it is not readily apparent whether factors underlying each of the UAE QTL detected in this study are genetic determinants of abnormal levels of urinary albumin indicative of impaired renal function or determinants of only small changes in baseline levels of albumin. The observed difference in UAE between the S and congenic strains are modest (15–40 mg/24 h). However, S and congenic strains were compared at an early age (~8 wk), when levels of UAE are relatively low in the S rat. The following question then arises: Are the allelic variants described in this study responsible for the substantial albuminuria and progressive renal lesions that ultimately develop in S rats as they age? Unfortunately, this question cannot be answered by the present data and will require future aging studies in congenic rats carrying individual QTL allelic variants. Nevertheless, the present linkage analysis did show that QTL for KLG mapped to most of the regions linked to UAE, demonstrating that even low levels of UAE (~10 mg/24 h) can be associated with overt, albeit early, kidney pathology.

The present study sought to identify QTL for several cardiovascular and renal phenotypes. The data demonstrated that factors such as age and diet can influence when and if a QTL is observed. Additionally, we clearly established that UAE QTL were present on RNO2, -6, -9, -11, and -13 by congenic strain analysis. Nevertheless, a significant amount of work is still required to further characterize each congenic strain and ultimately identify the underlying genetic factors responsible for susceptibility to renal damage in the S rat.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grant HL-066998 (to M. R. Garrett).


   ACKNOWLEDGMENTS
 
The authors thank Dr. John Rapp for the support and critical reading of this manuscript. The technical assistance by Hilary Good and Kris Farms is sincerely appreciated.


   FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: M. R. Garrett, Dept. of Physiology and Cardiovascular Genomics, Medical Univ. of Ohio, 3035 Arlington Ave., Toledo, OH 43614-5804 (e-mail: mgarrett{at}meduohio.edu).


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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