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Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODS AND MATERIALSRESULTSDISCUSSIONReferences |
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2,000 currently known rat genes in the renal medulla (a tissue critical for long-term blood pressure regulation) in SS and SS-13BN/Mcw rats in response to a high-salt diet (16 h, 3 days, or 2 wk). Differentially expressed genes in each between-group comparison were identified based on a threshold determined experimentally using a reference distribution that was constructed by comparing rats within the same group. A difference analysis of 54 microarrays identified 50 genes that exhibited the most distinct temporal patterns of expression between SS and SS-13BN/Mcw rats over the entire time course. Thirty of these genes could be linked to the regulation of arterial blood pressure or renal injury based on their known involvement in functional pathways such as renal tubular transport, metabolism of vasoactive substances, extracellular matrix formation, and apoptosis. Importantly, the majority of the 30 genes exhibited temporal expression patterns that would be expected to lower arterial pressure and reduce renal injury in SS-13BN/Mcw compared with SS rats. The phenotypic impact of the other 20 genes was less clear. These 50 genes are widely distributed on chromosome 13 and several other chromosomes. This suggested that primary genetic defects, although important, are unlikely to be solely responsible for the full manifestation of this type of hypertension and associated injury phenotypes. In summary, the results of this study identified a number of pathways potentially important for the amelioration of hypertension and renal injury in SS-13BN/Mcw rats, and these results generated a series of testable hypotheses related to the role of the renal medulla in the complex mechanism of salt-sensitive hypertension. microarray; 11ß-hydroxysteroid dehydrogenase; glucagon receptor; extracellular matrix; apoptosis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODS AND MATERIALSRESULTSDISCUSSIONReferences |
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This consomic strain can be used to develop congenic strains to identify small regions and eventually specific gene(s) on SS chromosome 13 that may contain primary genetic defects associated with blood pressure salt sensitivity. However, primary genetic defects, although very important, are unlikely to be solely responsible for the full manifestation of hypertension and associated injury phenotypes. These primary genetic defects could directly or indirectly affect the expression of genes across the genome. Functional pathways involving those genes could then importantly contribute to the development of salt-sensitive hypertension and associated injury. Due to the high degree of genetic identity and the substantial difference of blood pressure phenotypes, the comparison of SS-13BN/Mcw and SS provides an excellent tool with which to study these mechanistic pathways. To explore these pathways, a custom-made cDNA microarray representing 2,000 rat genes has been developed (13). These genes have been selected because most of them have some known biological functions, facilitating the effort focused on linking gene expression patterns with functional pathways. It also reduces the cost of these experiments, allowing studies with more replicates to be performed. It is important to note that only about 40 genes have been mapped to rat chromosome 13 (see Rat Genome Database, http://rgd.mcw.edu), and only 13 of them are included in this microarray. Therefore, this microarray is used primarily as a tool to study the mechanistic pathways of Dahl salt-sensitive hypertension including those involving genome-wide gene interactions, rather than identify the primary genetic defects on SS chromosome 13 that may initiate Dahl salt-sensitive hypertension.
In a previous study, we used this 2,000-gene microarray to examine gene expression profiles in the renal medulla (a tissue playing a critical role in the long-term regulation of arterial blood pressure; Refs. 5 and 9) in SS-13BN/Mcw and SS rats on either a low-salt diet or switched to a high-salt diet for 2 wk (13). It is known that SS rats exhibit a rapid reduction of medullary blood flow and develop hypertension rapidly and progressively when exposed to a high-salt diet (15). The present study was, therefore, designed to examine sequential changes of renal medullary gene expression in SS-13BN/Mcw and SS rats upon exposure to the high-salt diet for 16 h (overnight), 3 days, or 2 wk. These time points represented initial high-salt exposure without significant hypertension (16 h), the beginning of significant rise of arterial blood pressure (3 days), and established hypertension (2 wk) in SS rats (4, 15). Analysis of 54 microarrays identified 50 genes that exhibited the most distinct temporal patterns of expression between the two strains of rats over the time course studied. These expression patterns agreed well with the phenotypic differences between SS-13BN/Mcw and SS and suggested several functional pathways that might be important for the development of salt-sensitive hypertension and associated renal injury.
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METHODS AND MATERIALS |
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TOPABSTRACTINTRODUCTIONMETHODS AND MATERIALSRESULTSDISCUSSIONReferences |
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Dietary salt intake protocol and collection of tissue.
Nine SS rats and nine SS-13BN/Mcw rats were maintained on a normal light cycle (light from 6:00 AM to 6:00 PM) and on a low-salt (0.4% NaCl) diet until 11–12 wk of age. Three rats of each strain were then switched to a high-salt (4% NaCl) diet for 16 h (from 5:00 PM to 9:00 AM the next day), while another three rats switched to the high-salt diet for 3 days. On the day of tissue harvest, rats were anesthetized with Inactin (25 mg/kg ip) and ketamine (20 mg/kg im). Kidneys were removed quickly, and the renal medulla (both papilla and outer medulla) was selectively dissected using a sterile surgical blade and scissors, snap frozen in liquid nitrogen, and stored at -80°C. All tissue samples were collected between 9:00 AM and noon. Tissues were collected within 15 min following the administration of anesthesia. Therefore, anesthesia should have minimal confounding effects on gene expression, although they could not be completely ruled out.
Construction and hybridization of rat known-gene cDNA microarrays.
The description of microarray experiments in the present study conforms to the "minimum information about a microarray experiment" (MIAME) standard (2) to the extent possible. This includes detailed descriptions of custom-made microarrays, experimental design, hybridization protocols, and data analysis methods.
Custom-made cDNA microarrays containing cDNA probes that represented 80% of all currently known rat genes were used in the present study. The majority of these genes have some known biological functions in rats. These microarrays were constructed as we described previously (13) with the following modifications. cDNA probes for 1,871 genes, instead of 1,751 genes, were printed on this array, which included 1,687 clones purchased from Research Genetics (Huntsville, AL), and 184 genes cloned in our department or purchased from ATCC (Manassas, VA).
Microarrays were processed and hybridized using a two-color (Cy3 and Cy5) method with dye switching as we described previously (13).
Experimental design and overall analytical strategy.
Rats were paired for microarray hybridization, and the results were analyzed as shown in Fig. 1. Each of the four between-group comparisons comprised three pairs of rats examined by six microarrays with dye switching for each pair. Dye switching was not necessary for within-group comparisons, because the two rats of each pair were equivalent in terms of their treatment status. A total of 30 microarrays were hybridized in the present study. The expression data from the present study were combined with those from 24 microarrays hybridized in our previous study (13) and used to identify genes exhibiting the most distinct temporal patterns of expression between SS and SS-13BN/Mcw over the time course studied.
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A difference calculation was used to identify genes with the most distinct temporal patterns of expression between SS and SS-13BN/Mcw rats over the time course studied. This calculation measured the sum of the squared differences between the expression levels of each gene in the two rat strains at four time points (low-salt, high-salt 16 h, 3 days, and 2 wk), which is essentially a variant of the Euclidean distance calculation. Specifically, the expression level of a gene in SS rats on the low-salt diet (SSLS) was arbitrarily set at 0. The Ln(ratio) for this gene between each high-salt time point (16 h, 3 days, or 2 wk) and SSLS was used to indicate its relative expression level in SS rats at each high-salt time point. Relative expression levels of this gene in SS-13BN/Mcw rats at those four time points were similarly obtained with the expression level on the low-salt diet (13LS) set at 0. The difference of this gene’s expression pattern between SS and SS-13BN/Mcw rats over the time course was then calculated as D = 
4i = 1(Xi - Yi)2, in which Xi and Yi were the expression levels of this gene (obtained as described above) in SS and SS-13BN/Mcw rats, respectively, at the four time points. To take into consideration the fact that genes were not always expressed equally between SSLS and 13LS, relative expression levels in SS-13BN/Mcw were corrected by adding the Ln(ratio) between 13LS and SSLS. A second set of D values was calculated using these corrected expression levels. This second set of D values theoretically would better reflect the true expression differences. However, it had the disadvantage of being disproportionally affected by the single Ln(ratio) between 13LS and SSLS. Therefore, we used both sets of D values to rank the genes rather than relying on one of them. A gene was considered to exhibit highly distinct temporal patterns of expression between SS and SS-13BN/Mcw if its D values appeared in the largest 10% of both sets of D values and if it did not have any missing X or Y values over the entire time course. A cutoff at the largest 10% was chosen to yield a manageable set of genes. The validity of this cutoff level was supported by the fact that the majority of the genes identified were differentially expressed at least at one particular time point.
Northern blot.
mRNA expressions of 11 genes were further examined by Northern blotting as we described previously (13, 26).
Retrieval of gene mapping information.
Rat chromosomal locations of genes of interest were retrieved from the Rat Genome Database (RGD) maintained at our institution (http://rgd.mcw.edu) and the RGD Virtual Comparative Mapping Tool. This tool enables prediction of gene locations on the rat genome based on human or mouse genomes for those genes that were not directly mapped to the rat genome by genetic linkage or radiation hybrid methods.
Verification of clone identity.
Some of the genes identified as differentially expressed in the present study had been sequence-verified in our previous study (13). For the rest of the differentially expressed genes, PCR products were generated from corresponding clones and sequenced using the BDT chemistry (Applied Biosystems, Foster City, CA). Fifty-four partial sequences were successfully obtained and, based on BLAST searches, three (5.6%) did not match the identification provided by Research Genetics. These three clones were removed from the list of differentially expressed genes.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODS AND MATERIALSRESULTSDISCUSSIONReferences |
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Ln(ratio) values of the expression of 11 genes obtained from both microarrays and Northern blots are shown in Table 1. Ln(ratio) values from microarrays significantly correlated with those from Northern blots with a correlation coefficient of 0.376 (P = 0.01). Moreover, 9 of the 11 genes, with dipeptidyl peptidase and rat prolyl oligopeptidase (rPOP) as the exceptions, were consistently shown to be expressed either higher or lower in SS-13BN/Mcw compared with SS by both microarrays and Northern blots. In this and our previous (13, 27) studies, a total of 62 genes have been examined by both microarrays and Northern blots, and significant correlation between microarray and Northern blot results has been consistently observed. This represents one of the largest validation data sets in the microarray literature, and supports the overall reliability of the microarray technique we used.
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Extensive searches and evaluations of literature and publicly available databases were performed to retrieve information regarding the biological functions of proteins encoded by these 50 genes. Fifteen of these 50 genes, shown in Fig. 3, were found to be involved in the action or metabolism of substances that regulate renal tubular reabsorption and/or vascular tone. Examples of these substances included corticosterone, aldosterone, glucagon, natriuretic peptides, and arachidonic acid metabolites.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODS AND MATERIALSRESULTSDISCUSSIONReferences |
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Figure 6 depicts a hypothetical scheme summarizing several of these pathways that seem to be convincingly related to the amelioration of salt-sensitive hypertension and renal injury in SS-13BN/Mcw rats compared with SS rats, according to the known functions of those genes (1, 3, 6–8, 10–12, 16–18, 20, 21, 23–25). An interesting example is the 11ß-HSD pathway of glucocorticoid metabolism. In rats, 11ß-HSD type 1 has the ability to convert inert 11-dehydrocorticosterone (functionally similar to cortisone in humans) to active corticosterone (functionally similar to cortisol in humans), whereas type 2 inactivates corticosterone back to 11-dehydrocorticosterone. Cortisol is a potent stimulator of mineralocorticoid receptors located in the distal nephron (7), leading to sodium retention. Homozygous loss-of-function mutations in the 11ß-HSD type 2 gene were found to cause the human syndrome of apparent mineralocorticoid excess characterized with hypertension (16). The downregulation of 11ß-HSD type 1 and the upregulation of 11ß-HSD type 2 in SS-13BN/Mcw compared with SS shown in the present study, therefore, would be expected to decrease the level of active glucocorticoids in the kidney of SS-13BN/Mcw rats. This is consistent with lower arterial blood pressure observed in SS-13BN/Mcw compared with SS (4). Specific hypotheses could be developed to test the functional importance of this and many other pathways (Figs. 3–6) in Dahl salt-sensitive hypertension.
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The fact that only a few of the 50 genes with distinct temporal patterns of expression were mapped to rat chromosome 13 is reminiscent of the results of our previous study (13). This could be caused by several mechanisms. Genomic differences on chromosome 13 and the products they encode could be regulatory factors that can directly influence the expression of genes on many chromosomes. These genome-wide gene interactions could also be indirect through downstream signaling pathways or as a result of feedback regulation by functional alterations. In addition, the possibility of residual BN alleles on chromosomes other than chromosome 13 in SS-13BN/Mcw rats cannot be excluded. It remains to be determined whether the differentially expressed genes that did map to chromosome 13 in the present study indeed contain fundamental genetic differences associated with blood pressure salt sensitivity. Approaches such as generating congenic lines and sublines, gene targeting or transfer, and mutation identification will be useful in this regard (19). In the meantime, multiple pathways identified in the present study, whether located on chromosome 13 or not, should improve our understanding of the complex network of pathways underlying the pathophysiology of Dahl salt-sensitive hypertension. The importance of these genes is supported not only by the theoretical presence of such a network of pathways but also by the fact that the expression patterns of the majority of these genes were consistent with the blood pressure and injury phenotypes in these two strains of rats. This is interesting, particularly because it is not unusual for microarray studies to generate expression profiles that are difficult to reconcile with known phenotypes.
A novel "reference distribution" method was used to determine the threshold of differential expression in the present study. With this method, an apparent difference between two groups must exceed the random variation between individual samples in each group to be considered a true difference. This, indeed, is a common principle behind most statistical tests in biological and other fields of investigation. The ratio distributions for between-group comparisons had consistently larger standard deviations than the reference distribution, indicating that the magnitude of between-group differences in mRNA expression exceeded that of random variations. With the 99.9% interval used in the present study, not more than 1 gene would be expected to be identified as differentially expressed by chance alone. If one would like to reduce the number of false negatives while tolerating a larger number of false positives, then the threshold could be reduced, e.g., to a value corresponding to a 95% interval of the reference distribution, knowing that they would now have 50 (5% of 1,000) false-positive genes.
In summary, cDNA microarrays with newly developed analytical methods have been used to examine sequential changes of renal medullary gene expressions in SS and consomic SS-13BN/Mcw rats. The results identified a number of pathways potentially important for the amelioration of hypertension and renal injury in SS-13BN/Mcw rats and generated a series of testable hypotheses related to the role of the renal medulla in the complex mechanism of salt-sensitive hypertension. These results also indicated that multiple pathways could be involved in the development of Dahl salt-sensitive hypertension and suggested that primary genetic defects, although important, are unlikely to be solely responsible for the full manifestation of this type of hypertension and associated injury phenotypes.
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ACKNOWLEDGMENTS |
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This study was supported by National Heart, Lung, and Blood Institute Grants HL-66579, HL-54998, and HL-29587.
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. Liang, Dept. of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226 (E-mail: mliang{at}mcw.edu).
10.1152/physiolgenomics.00089.2002.
1 The Supplementary Material for this article is available online at http://physiolgenomics.physiology.org/cgi/content/full/12/3/229/DC1. 
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References |
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TOPABSTRACTINTRODUCTIONMETHODS AND MATERIALSRESULTSDISCUSSIONReferences |
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). LS, maintained on the low-salt diet; HS, switched to the high-salt diet for the time period indicated. Ln(ratio) values shown are between each condition and SS on the low-salt diet. Temporal expression patterns of the majority of these genes are consistent with the lower blood pressure seen in SS-13BN/Mcw compared with SS when both strains of rats are exposed to the high-salt diet. rPOP, rat prolyl oligopeptidase.
, upregulation or increases in SS-13BN/Mcw compared with SS; 
, downregulation or decreases in SS-13BN/Mcw compared with SS.