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Physiol. Genomics 18: 218-225, 2004. First published May 25, 2004; doi:10.1152/physiolgenomics.00068.2004
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Received 19 March 2004; accepted in final form 21 May 2004.
Physiological Genomics 18:218-225 (2004)
1094-8341/04 $5.00 © 2004 American Physiological Society

Genetic linkage of albuminuria and renal injury in Dahl salt-sensitive rats on a high-salt diet: comparison with spontaneously hypertensive rats

Anja-Kristin Siegel1, Peter Kossmehl1, Michael Planert1, Angela Schulz1, Markus Wehland1, Monika Stoll2, Jan A. Bruijn3, Emile de Heer3 and Reinhold Kreutz1,4

1 Institut für Klinische Pharmakologie und Toxikologie, Campus Benjamin Franklin, Charité Universitätsmedizin Berlin, Berlin, Germany
4 Medizinische Klinik mit Schwerpunkt Nephrologie und Internistische Intensivmedizin, Campus Virchow-Klinikum, Charité Universitätsmedizin Berlin, Berlin, Germany
2 Institut für Arterioskleroseforschung, Westfälische-Wilhelms-Universität Münster, Münster, Germany
3 Department of Pathology, Leiden University Medical Center, 2300 RC, Leiden, The Netherlands


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Our aim was to study the effects of high-salt diet on the genetics of albuminuria and renal injury in the Dahl salt-sensitive (SS) rat. We compared SS with salt-resistant spontaneously hypertensive rats (SHR) and with genetically related salt-sensitive stroke-prone SHR (SHRSP). Moreover, we performed genome-wide linkage analysis to identify quantitative trait loci (QTL) contributing to salt-induced renal injury in an F2 population derived from SS and SHR (n = 230). In response to high-salt diet SS and SHRSP developed a striking increase in systolic blood pressure, urinary albumin excretion (UAE), and renal damage indices compared with SHR. Both SHRSP and SS developed severe glomerulosclerosis, whereas microangiopathy, tubulointerstitial fibrosis, and inflammation were more pronounced in SHRSP. We detected two QTL with significant linkage to UAE on rat chromosomes (RNO) 6 and 19. Comparison with the recently identified salt-independent UAE QTL in young animals revealed that the UAE QTL on RNO6 is unique to high-salt conditions, whereas RNO19 plays a significant role during both low- and high-salt conditions. Some F2 animals demonstrated severe microangiopathy and tubulointerstitial injury, which exceeded the degree observed in the parental SS strain. Three loci demonstrated suggestive linkage to these phenotypes on RNO3, RNO5, and RNO20, whereas no linkage to glomerular damage was found. Further analyses at these loci indicated that the severity of renal injury was attributable to the SHR allele. Our data suggest that the SHR genetic background confers greater susceptibility for the development of microangiopathy and tubulointerstitial injury in salt-sensitive hypertension than the SS background.

quantitative trait loci; hypertension


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
SALT-SENSITIVE HYPERTENSION plays a significant role as a factor contributing to the manifestation and progression of cardiovascular and chronic renal diseases (2, 12, 13, 19). It is a well-recognized clinical phenomenon that subgroups of patients with essential hypertension exhibit salt sensitivity and more severe progression of hypertensive target-organ damage over time (2, 20, 32, 33). Familial aggregation and the higher prevalence of salt-sensitive hypertension in specific ethnic populations (17, 20, 31) point to the potential importance of genetic factors. This is also supported by several genetic rat models that display salt-sensitive hypertension and related target-organ damage as an inherited trait, thus representing an attractive substitute for the investigation of the polygenetic basis of the human disease (10, 24).

We have recently shown, that the Dahl salt-sensitive (SS) rat when raised on a low-sodium diet demonstrates not only spontaneous hypertension but develops also at 4 wk of age a significant increase of urinary albumin excretion (UAE) (22). In addition, two independent linkage studies demonstrated that several quantitative trait loci (QTL) are linked to the development of increased UAE in the SS rat on a low-sodium diet (5, 22). The first aim of the current study was therefore to study the effect of high-salt diet on the genetics of UAE in the SS rat and to test whether UAE QTL are unique to either low- or high-salt diet conditions. The second goal was to explore the genetic basis underlying the salt-induced renal tissue damage in the SS rat. We therefore compared the effects of high-salt loading on renal histopathology findings between the SS strain and other rat strains that also demonstrate spontaneous hypertension, i.e., the salt-resistant spontaneously hypertensive rat (SHR) (25) and the salt-sensitive stroke-prone spontaneously hypertensive rat (SHRSP) (25) strains. In addition, we performed a genome-wide genetic linkage and QTL mapping analysis in F2 progeny derived from SS and the contrasting SHR strain in response to the high-salt diet. Hence, in a previous study we have shown that the SHR strain is resistant to salt-induced systolic blood pressure (SBP) increases and renal disease progression even after unilateral nephrectomy (25). Therefore, it seems of interest to study the genetic basis of salt-dependent increase of UAE and renal glomerular and interstitial injury in an experimental cross between the susceptible SS and the resistant SHR strain, which has not been done so far. In particular, here we report for the first time a detailed cosegregation analysis of glomerular and tubulointerstitial injury including fibrosis, inflammation, and microangiopathy between the SS and SHR strains.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals.
All animals studied were males and were obtained from our colonies at the Freie Universität Berlin, Benjamin Franklin Campus as reported (25, 29). Recently, we were asked to register our colonies at the Institute for Laboratory Animal Research (http://dels.nas.edu/ilar/), where the laboratory code "Rkb" was assigned. In addition to the SHR/Rkb and SS/Rkb strains, we studied parental animals from our SHRSP rat strain (SHRSP/Rkb) (25). For linkage analysis we generated an (SS x SHR)F2 cross population including 230 male animals. The genetic analysis of cardiovascular hypertrophy in this population was recently reported elsewhere (29).

Determination of phenotypes.
One group of each parental strain (n = 10–12, respectively) was studied under a normal diet containing a low content of salt, 0.2% NaCl by weight. Salt loading with a diet containing 4% salt by weigh was performed in additional groups of parental animals of each strain (n = 10–12, respectively) and in all F2 animals following a standardized protocol (29). In brief, at the age of 6 wk animals received a 4% salt (by weight) diet (Ssniff, Soest, Germany) for 8 wk. SBP was then measured at the age of 14 wk in awake animals by the tail-cuff method, which has been previously validated and reported (14, 29). In brief, two training sessions were performed on two separate days. Subsequently, the final blood pressure measurements were recorded on the three consecutive days. Because of three sets of two measurements at each session, the individual blood pressure phenotype was based on a maximum of 18 measurements for each rat. A minimum of 12 measurements was required for inclusion in the analysis, which was achieved in all 230 animals.

For urine analysis rats were placed into metabolic cages for 24 h for adaptation, and urine was subsequently collected over an additional 24-h period. Albumin concentrations were measured by a sensitive and rat-specific ELISA technique established in our laboratory using a rat-specific antibody (ICN Biomedicals, Eschwege, Germany) (14). Urinary protein excretion (UPE) was determined by the Bradford method. Subsequently, animals were killed under ether anesthesia at 15 wk. The spleen and both kidneys were excised. The body and total kidney weights were determined. For light microscopy evaluation a midcoronal section of the left kidney was immersed in Dubosq-Brasil solution and embedded in paraffin for histological studies as reported (26). Samples were cut into 3-µm sections and stained with periodic acid-Schiff reagent (PAS) followed by hematoxylin counterstaining. All sections were first analyzed by semiquantitative histological grading (0 = absent, 1+ = mild, 2+ = moderate, and 3+ = severe) for the severity of focal segmental glomerulosclerosis, tubulointerstitial infiltration, and microangiopathy. In addition, the glomerulosclerosis index (GSI) (23) and the amount of renal interstitial fibrosis (RIF) was determined after Sirius red staining (18). For GSI, 20 fields per kidney were examined, and lesions were graded as follows: grade 0, no changes; grade 1, lesion involving less than 25%; grade 2, lesion affecting 25–50%; and grade 3, lesion involving more than 50% of the field. The resulting index in each animal was expressed as mean value of all scores obtained. All injury parameters were assessed independently by two investigators in a blinded manner. Quantification of RIF was performed with use of a video camera combined with a video control system (Sony model MC-3255; AVT Horn, Aalen, Germany) adapted to a Zeiss Axiophot microscope. Image analysis was performed with the use of freely available software (Scion Image 1.62a, Scion) 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 given as percent was determined; 10 sections per animal were averaged to obtain individual RIF phenotypes for each rat.

Linkage analysis, QTL mapping, and statistical analysis.
A complete genome screen was performed as previously described (26), and linkage with 210 polymorphic microsatellite markers was performed as reported (29).

Prior to linkage analysis, phenotypic distribution was tested using the Kolmogorov-Smirnov Test to assure normal distribution of the trait within the F2 population, as required for parametric linkage analysis. Traits failing the requirements were transformed using either a logarithmic or square root transformation and retested for normalcy (30). All parameters including GSI and RIF, except the semiquantitative renal injury scores, could be analyzed by parametric linkage analysis. QTL mapping was performed with the MAPMAKER/QTL software (16). Renal injury scores for focal segmental sclerosis, interstitial inflammation, and microangiopathy were additionally analyzed by nonparametric Kruskal-Wallis test. The threshold for significant linkage was set to a LOD score of 4.3 or P = 0.000052 and for suggestive linkage to a LOD score of 2.8 or P = 0.0016, as recommended (15).

All data are expressed as means and SD. Genotype-phenotype correlation was performed at genetic markers closest to the QTL peak using one-way analysis of variance (ANOVA) followed by post hoc test (Bonferroni). In addition, the Kruskal-Wallis test was used as applicable. Except for linkage results, differences were considered significant at the level of P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Phenotypes in parental strains.
The data for SBP and UAE in parental strains are presented in Fig. 1. Under a low-salt diet all three strains showed similar SBP values (Fig. 1A). After high-salt diet, SBP values did not significantly change in the SHR strain, whereas both SS and SHRSP animals developed a similar pronounced increase in SBP compared with SHR (P < 0.001) and their corresponding low-salt diet group (P < 0.001, respectively). In contrast to SBP, UAE levels showed already a significant difference between strains under low-salt diet (Fig. 1B). That is, under low-salt diet, UAE levels were already significantly elevated in the SS strain compared with SHR and SHRSP (P < 0.0001, respectively). High salt led to a marked elevation of UAE in SHRSP and a further increase of UAE in SS rats compared with their corresponding low-NaCl values (P < 0.001, respectively). After high-NaCl diet, the SHRSP strain reached UAE levels that were similar to the low-NaCl values in the SS strain (Fig. 1B). SHR animals demonstrated no significant change in response to high-NaCl diet.



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  		Fig. 1. Systolic blood pressure (SBP; A) and urinary albumin excretion (UAE; B) in adult spontaneously hypertensive rats (SHR), stroke-prone spontaneously hypertensive rats (SHRSP), and salt-sensitive Dahl rats (SS) after normal salt diet containing 0.2% NaCl by weight (open bars) and high-salt diet containing 4% NaCl by weight (solid bars). *P < 0.001 vs. SHR; #P < 0.01 vs. SHRSP; §P < 0.001 vs. SHR and SHRSP after normal salt diet, respectively.

 
Histology analysis by light microscopy in the three parental strains under a low-NaCl diet demonstrated no severe renal abnormalities (Figs. 2 and 3). On the high-salt diet SHR animals developed a low degree of focal segmental glomerulosclerosis without marked tubulointerstitial or vascular abnormalities (Figs. 2B and 3B, respectively). Both SHRSP and SS developed severe glomerulosclerosis on the high-salt diet (Fig. 2, D and E, and Fig. 3, D and E, respectively), whereas the SHRSP strain developed clearly more severe microangiopathy, tubulointerstitial infiltration, and fibrosis.



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  		Fig. 2. Histopathology of the kidney in hypertensive rat strains at 15 wk of age, respectively. Paraffin sections were stained with periodic acid-Schiff reagent (PAS). A: SHR at normal salt diet virtually without severe renal abnormalities (V, vein; G glomeruli). B: SHR kidney section after high-salt diet. Note the development of focal segmental glomerulosclerosis without marked tubulointerstitial, or vascular abnormalities (SS animals demonstrated a similar picture, not shown). C: SHRSP at low-salt diet. Note the glomeruli (G) and arteries (A) without abnormalities. D: SHRSP at high-salt diet. Note the development of severe microangiopathy in renal arteries (A), tubulointerstitial fibrosis, and glomerulosclerosis (G). E: SS at a high-salt diet. Note the marked hypertrophy and glomerular collapse (G), tubulointerstitial fibrosis (*), absence of microangiopathy (A), and tubulointerstitial infiltration with inflammatory cells. F: F2 cross rat derived from SS x SHR. Note the marked hypertrophy and severe glomerulosclerosis (G), tubulointerstitial fibrosis, microangiopathy (A), and tubulointerstitial infiltration with inflammatory cells. Original magnification x200.

 


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  		Fig. 3. Histopathology of glomeruli from hypertensive rat strains at 15 wk of age, respectively. Paraffin sections were stained with PAS. A: spontaneous hypertensive rat (SHR) at normal salt diet virtually without glomerular abnormalities. B: glomerulus from SHR after high-salt diet with hilar glomerular thrombosis. C: glomerulus from SHRSP at low-salt diet without glomerular abnormalities. D: glomerulus from SHRSP at high-salt diet with segmental glomerular thrombosis and collapse (G). E: glomerulus from SS rat at a high-salt diet. Note the marked hypertrophy and glomerular, PAS-positive extracellular matrix expansion. F: F2 cross rat derived from SS x SHR. Note the marked hypertrophy and severe endothelial damage with microthrombi and influx of inflammatory cells. Original magnification x400.

 
Cosegregation and QTL mapping analysis in F2 animals.
The ranges of minimal and maximal SBP (149.5–267.7 mmHg) and UAE (0.31–288 mg/24 h) levels observed in the F2 population were in agreement with the contrasting phenotypes of the parental SHR and SS strains. Correlation analysis revealed a significant correlation between SBP and UAE (r = 0.39, P = <0.0001). Histology analysis in the F2 animals demonstrated overall relatively low mean values after grading for renal injury scores. The mean GSI was 1.64% (SD 0.34), and the mean RIF score was 6.52% (SD 2.16). However, some F2 animals demonstrated severe glomerulosclerosis and pronounced tubulointerstitial injury and microangiopathy (Figs. 2F and 3F, respectively). The latter was in contrast to the parental SS strain, which exhibited on the high-salt diet only a mild degree of tubulointerstitial damage and microangiopathy (Figs. 2E and 3E, respectively). UAE but not SBP showed a weak correlation with the GSI (r = 0.16, P = 0.01), whereas both parameters exhibited no correlation with RIF. SBP and UAE were significantly correlated with tubulointerstitial inflammation (r = 0.44 and r = 0.46, P < 0.0001, respectively) and microangiopathy (r = 0.29 and r = 0.45, P < 0.0001, respectively), and a strong correlation was observed between both injury parameters (r = 0.62, P < 0.0001).

Suggestive or significant QTL with linkage to SBP, UAE, or UPE were identified on RNO1, RNO3, RNO6, RNO8, RNO9, and RNO19 (as summarized in Fig. 4). Only one significant QTL with a LOD score >4.3 was found for SBP on RNO9 as reported (29), and two significant QTL were identified with linkage to both UAE and UPE on RNO6 and RNO19, respectively. In our recent analysis we identified seven suggestive or significant UAE QTL on RNO2, RNO6, RNO8, RNO9, RNO10, RNO11, and RNO19 that contributed to the genetic determination of early onset albuminuria in young animals at 8 wk of age (22). The comparison with the current study on high-salt diet in older animals revealed that only the QTL on RNO19 exhibited significant linkage in both studies. Indeed, direct visual comparison of the LOD plots demonstrated almost identical linkage results in both young animals on low-salt diet and in old animals on a high-salt diet on RNO19 (Fig. 5). At this QTL on RNO19, the SS allele caused an increase of UAE in both crosses as expected from the parental data. Heterozygous animals showed a similar increase of UAE [24.6 (SD 56.0) mg/24 h] as animals carrying two SS alleles [28.1 (SD 44.3) mg/24 h] compared with animals carrying two SHR alleles [5.2 (SD 5.4) mg/24 h; LOD = 5.46]. Further comparison of LOD plots showed the decrease of statistical linkage on RNO2 and RNO9 compared with the significant linkage results in the young low-salt animals (Fig. 5). Interestingly, the analysis on RNO6 showed the decrease of linkage at marker D6Rat12 in the salt-loaded F2 animals compared with the low-salt young animals, whereas a new second QTL at the other end of the chromosome was significantly linked to UAE only in the F2 animals after high-salt loading (LOD = 4.47, Fig. 5). In contrast to the QTL on RNO19, however, the increase in UAE and UPE (LOD = 8.83) at the QTL on RNO6 at marker D6Rat69 was related to the presence of the homozygous SHR/SHR genotype, which led to an approximate twofold increase of UAE and UPE compared with heterozygous and F2 animals carrying two SS alleles.



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  		Fig. 4. Chromosomal maps for rat chromosomes (RNO) 1, 3, 6, 8, 9, and 19 demonstrating genetic linkage to SBP, UAE, or urinary protein excretion (UPE). Chromosomal markers are given on the left of each chromosome and ordered according to the linkage map at distances of centimorgans (cM). A genetic distance of 10 cM is indicated. Vertical lines at the right of each chromosome represent the 1-LOD interval or 95% confidence interval for placement of the QTL either due to significant linkage (solid lines) or suggestive linkage (broken lines). The small horizontal crossing line indicates the linkage peak. The margins of these intervals in relation to the chromosomal position are additionally indicated by horizontal dotted lines.

 


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  		Fig. 5. Chromosomal maps and LOD plots for rat chromosomes (RNO) RNO2, RNO6, RNO9, and RNO19, which demonstrated significant linkage (LOD > 4.3) to early onset albuminuria in our recent study in animals raised on a low-sodium diet as indicated with the thin curve (12). The LOD plots for the current study in older animals studied on high-salt diet are indicated by the thick curve. Chromosomal markers are given at the bottom of each chromosome and ordered according to the linkage map at distances of centimorgans (cM). A genetic distance of 10 cM is indicated. The horizontal dotted line at LOD = 2.8 indicates the threshold for suggestive linkage, and the solid line at LOD = 4.3 indicates the threshold for significant linkage.

 
The analysis for renal injury parameters demonstrated no linkage to the glomerular damage parameters (GSI and semiquantitative grading for focal segmental sclerosis), whereas suggestive linkage was found to tubulointerstitial injury parameters and to microangiopathy. At D3Mgh9 we detected suggestive linkage slightly below the significant linkage threshold (P = 0.000085) to tubulointerstitial inflammation grading. At this locus on RNO3, it appeared that interstitial inflammation was absent, i.e., relating to a score of 0, in all 58 F2 animals carrying the homozygous SS/SS genotype. Thus the manifestation of interstitial inflammation depended on the presence of the SHR allele at this locus. Moreover, the SHR allele at D3Mgh9 also demonstrated suggestive linkage to increased SBP (P = 0.0045) and UPE (P = 0.011, Fig. 4). The SHR allele at this locus on RNO3 also exhibited a weak effect on microangiopathy grading (P = 0.028). Suggestive linkage to histology grading for microangiopathy was detected on RNO5 at D5Mit10 (P = 0.0014). Homozygosity for the SHR allele was also linked to more severe damage of microvessels, and the SHR allele at this locus on RNO5 exhibited also a weaker effect on interstitial inflammation grading (P = 0.069).

QTL mapping for RIF revealed suggestive linkage on RNO20 at marker D20Rat12 (LOD 2.93, P = 0.0011), where the amount of RIF was also higher in F2 animals carrying the SHR/SHR genotype. This locus, however, demonstrated no linkage to SBP, UAE, UPE, or other renal injury parameters.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The genetics of salt-sensitive blood pressure regulation in the SS model has previously been extensively studied (24). More recently, two blood pressure QTL mapping studies were performed between SS rats and models with spontaneous hypertension, and blood pressure QTL were mapped to RNO3, RNO8, and RNO9 (6, 7). In these experiments F2 rats were exposed to a high-salt diet containing 8% NaCl, and data for UAE or renal damage were not included.

In the current study we mapped suggestive or significant blood pressure QTL on RNO1, RNO3, RNO6, and RNO9 (29) and identified a colocalization with UAE or UPE QTL on RNO3, RNO6, and RNO9, with the latter suggesting that the linkage to UAE and UPE is a consequence of a blood pressure effect. In our recent report we studied SS rats that were raised on a low-sodium diet and demonstrated that early onset of albuminuria in SS rats takes place before the onset of ultrastructural glomerular changes (22). Moreover, linkage analysis in experimental crosses between SS and SHR rats under low-sodium dietary conditions indicated a polygenetic determination of UAE in the SS strain by the interaction of 7–10 UAE QTL (5, 22). The comparison of our previous data with the current results revealed that only the UAE QTL on RNO19 contributed also with significant linkage to the progression of UAE and UPE in response to high-sodium diet in older animals (22).

The SS allele caused an increase of UAE at the QTL on RNO19 in both crosses as expected from the parental data. In contrast to the recent study in young animals in which the SS allele demonstrated a recessive effect with a trend toward a codominant effect on UAE (22), in the current study with salt-loaded animals a dominant effect of the SS allele on UAE and UPE was observed.

This is in contrast to previous findings in rat models (5, 21, 22, 26, 28) and in human studies (3, 4) which indicated collectively that UAE or UPE is largely determined by recessive genetic effects. As previously reported the overall genetic effect of UAE in the cross between SS and SHR when studied under low-sodium diet is recessive, since F1 animals demonstrated UAE levels similar to SHR (12). Although we have not studied the effects of high-salt loading in F1 animals, the relevance of the UAE QTL on RNO19 and its potential dominant effect should be tested in future experiments when congenic and consomic strains for this locus and RNO19 derived from SHR and SS will be available. These studies will also take advantage of the database provided by the consomic rat development and phenotyping project (http://pga.mcw.edu/), where physiological data including renal damage in consomic strains involving SS and normotensive controls are available.

Moreover, the QTL region on RNO19 is not linked to SBP but demonstrated in our recent analysis additional linkage to cardiovascular hypertrophy parameters (29). Taken together, these data suggest the existence of one or more QTL on RNO19 that influence the manifestation of both cardiovascular and renal target organ damage in salt-sensitive hypertension.

In contrast to the QTL on RNO19, the increase in UAE and UPE at the QTL on RNO6 was related to a recessive genetic effect that was conferred by the SHR allele. The comparison with the linkage results in young animals without salt loading indicated that there are two UAE QTL on either end of the chromosome with different effects on early UAE in young low-salt animals and in older animals after salt loading (22). Thus the UAE QTL on RNO6 at D6Rat69 with significant linkage to both UAE (LOD 4.47) and UPE (LOD 8.83) was unique to the current high-salt study. However, our results are in agreement with the study by Garrett et al. (5), who also demonstrated linkage to two independent UAE QTL on RNO6, termed QTL1 and QTL2. In their study, the QTL1 was not detectable in young animals at 8 wk of age but demonstrated significant and suggestive linkage at 12 and 16 wk of age, respectively. However, since Garrett et al. (5) performed time-course analysis in F2 animals on a low-salt diet, the increase of linkage at their QTL1, which overlaps with the strong and significant QTL at D6Rat69 of the current cross, suggests that age is more relevant than salt loading for the increasing linkage found at this QTL in this report. The additional suggestive linkage to SBP at this locus on RNO6 points to the relevance of a hemodynamic effect mediating the linkage to UAE and UPE. Nevertheless, this QTL seems to be specific for the determination of renal target organ damage, since we observed no linkage to cardiovascular hypertrophy parameters at this QTL (29).

In the recent linkage study between SS and SHR, Garrett and coauthors (5) could show that most identified UAE QTL colocalized with QTL for kidney lesions under low-salt diet. In this study, renal damage was assessed by a grading system in which changes in glomeruli, tubules, and vasculature were integrated into one kidney lesion score without differentiation between compartments (5). In contrast, in the current study we set out to determine the distinct effects of salt loading on glomerular, tubulointerstitial, and microvascular injury. No significant linkage was found for glomerular damage, suggesting that there are no major genetic factors contributing to glomerular structural injury in the F2 hybrids. On the other hand this finding can be also explained by the fact that the percentage of sclerotic glomeruli in the F2 animals was overall very low. Moreover, in previous three-dimensional reconstructions of focal glomerular lesions, we have shown that focal lesions can easily be underscored (9).

A suggestive QTL on RNO20 was linked to the amount of RIF as determined by Sirius red staining. As previously shown by Grimm et al. (8), morphometric analysis of Sirius red surface area was more sensitive for differential scoring of tubulointerstitial fibrosis than any other histological grading. In contrast to our recent findings in the Munich-Wistar-Frömter rat, in which a QTL on RNO6 is linked to both early onset albuminuria and RIF (27), the locus on RNO20 identified here is not significantly linked to UAE, UPE, or any other renal damage index.

Two potential QTL on RNO3 and RNO5 predisposed for tubulointerstitial infiltration with inflammatory cells in the F2 population. This was of interest, since only a mild degree of renal interstitial diseases and microangiopathy was noted in the parental SS strain. In addition, these two loci predisposed also for the development of microangiopathies. Our comparison of the parental SS and SHRSP strains demonstrates that there is considerable variation in the severity of microangiopathy and tubulointerstitial disease in animals with severe salt-sensitive hypertension with mild or almost absent lesions in the SS and more severe damage in the SHRSP strain in the face of similar blood pressures. The finding of mild focal, tubulointerstitial damage in the SS rat is in agreement with a previous study in which the development of tubulointerstitial injury in SS rats (obtained from an inbred colony at the Medical College of Wisconsin) was observed between 3 and 9 wk of age compared with the Dahl salt-resistant (SR) strain (11). In this study, however, the development of tubulointerstitial injury may have been even aggravated by the secondary activation of the renin-angiotensin system (RAS) in response to a low-salt diet, since animals were raised under a low-sodium diet containing only 0.1% NaCl (11). Hence, in a rat model of cyclosporin nephropathy it was shown that microvascular and tubulointerstitial disease occurs only under low-salt dietary conditions (1, 12). Our finding that some F2 animals demonstrated pronounced renal inflammation, interstitial fibrosis, and microangiopathy which exceeded the degree observed in the parental SS strain could be explained by the genotype-phenotype analysis at the loci on RNO3 and RNO5. This analysis revealed that the manifestation and severity of microangiopathy and interstitial inflammation were related to the presence of the SHR allele. Although a trend for increased interstitial infiltration depending on the presence of the SS allele was found on RNO19, the detection of F2 animals with microangiopathy and interstitial inflammation seems to be largely attributable to the SHR genetic background. It appears possible that tubulointerstitial infiltration may be causally linked to hypertension-induced microangiopathy, since the latter may precede the former. As recently pointed out by Johnson et al. (12), ischemia of tubules may lead to upregulation of leukocyte adhesion molecules and infiltration of mononuclear cells.

Between the parental SS and SHRSP strains studied under high-salt diet, we observed no significant blood pressure difference. Consequently, there was no significant correlation between renal injury parameters and tail-cuff blood pressures in parental strains under these conditions. This suggests that blood pressure-independent, i.e., genetic, effects might contribute to the difference observed particularly for tubulointerstitial inflammation and microangiopathy between SS and SHRSP. This notion is supported by the finding on RNO3 where, in agreement with the parental SS data, tubulointerstitial inflammation was absent in all 58 F2 animals carrying the homozygous SS/SS genotype. A limitation of the current study is, however, the use of tail-cuff blood measurement and only one time point analysis for blood pressure phenotyping. Consequently, we cannot exclude the possibility that blood pressure effects over time or differences between nocturnal and daytime pressures could have contributed to differences in renal injury patterns in parental strains and individual F2 animals. Nevertheless, in the F2 animals we observed a significant correlation between SBP and microangiopathy and tubulointerstitial inflammation such that 19% and 8% of the variance of these phenotypes can be explained by blood pressure variation, whereas GSI and RIF demonstrated no correlation with blood pressures. Clearly, further pursuit in congenic or consomic strains is necessary to further confirm and dissect our linkage results between SS and SHR on albuminuria and renal injury parameters and to evaluate the relevance of blood pressure-independent effects.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by grants from the Deutsche Forschungsgemeinschaft (KR 1152/2-1, GK 462) and the Bundesministerium für Bildung und Forschung (Nationales Genomforschungsnetz: HK-Berlin 01GS0106/01GS0156).


   ACKNOWLEDGMENTS
 
We acknowledge the contributions of Heidelinde Müller, Gabriele Siebert, and Bettina Lack for excellent laboratory assistance.


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

Address for reprint requests and other correspondence: R. Kreutz, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Freie Universität Berlin, Hindenburgdamm 30, 12200 Berlin, Germany (E-mail: Kreutz{at}medizin.fu-berlin.de).

10.1152/physiolgenomics.00068.2004


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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