Serial analysis of gene expression in mouse kidney following angiotensin II administration

Faina Schwartz, Arvi Duka, Elena Triantafyllidi, Conrado Johns, Irena Duka, Jing Cui and Haralambos Gavras

Department of Medicine, Hypertension Section, Boston University School of Medicine, Boston, Massachusetts 02118


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
As a new line of inquiry into the molecular mechanisms underlying pathophysiological processes associated with angiotensin (ANG II)-dependent hypertension, we applied the method of serial analysis of gene expression (SAGE) to examine genome-wide transcription changes in the kidneys of mice that developed hypertension in response to chronic ANG II administration. Mice were infused subcutaneously via osmotic minipumps with ANG II for 7 days, and systolic blood pressure was measured by tail-cuff plethysmography. Subsequently, mice were euthanized, and the total RNA isolated from the kidneys was used to construct SAGE libraries. Comparison of 11,447 SAGE tags from the hypertensive kidneys, representing 5,740 unique transcripts, and 11,273 tags from the control kidneys, corresponding to 5,619 different transcripts, identified genes that are significantly (P < 0.05) down- or upregulated in the hypertensive kidney. Our assessment of the genome-wide influence of ANG II resulted in the detection of several novel genes and in a recognition of potential new roles for the previously characterized genes, thus providing new probes with which to further explore the ANG II effects in normal and disease states.

hypertension; mouse kidney transcriptome; angiotensin excess; tissue kallikrein; cathepsin D


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE COMPLEXITY OF COMMON HYPERTENSION (HTN), a polygenic and multifactorial disorder, is confounded by extreme heterogeneity, as underlying intrinsic and environmental factors vary between individuals and populations (30). Not surprisingly, inquiries into genetic causes of essential HTN, such as genome scans, linkage and association studies, have produced inconsistent findings (13, 28). Differential gene expression profiling provides an alternative and complementary approach to genomics for elucidation of the molecular chain of events leading to HTN. As blood pressure (BP) homeostasis involves complex interplay between vascular, renal, endocrine, and nervous functions, studies focusing on one gene or on the components of one biochemical pathway, based on our present understanding of physiology, are often difficult to extrapolate to a true physiological in vivo milieu. In contrast, genome-level transcriptional profiling of a given cell, tissue, or organ provides a map of interconnected molecular pathways and reflects coordinated response of their various components to a given physiological condition.

We used the technique of serial analysis of gene expression (SAGE) (63) to probe global transcription changes in mice with ANG II-induced HTN. The renin-angiotensin-aldosterone system has been extensively studied for more than a century (40). ANG II exerts a plethora of effects on several target organs, including blood vessels, kidney, adrenal, and heart, and influences many physiological functions, most notably BP and fluid and electrolyte homeostasis (11). In animal models, administration of exogenous ANG II, in addition to its effect on BP, is known to cause necrotic cardiac, arterial, and renal lesions (18), inhibit fibrinolysis (61), stimulate formation of reactive oxygen species (69), and induce apoptosis (15). Endogenous ANG II excess plays a key role in HTN, congestive heart failure, and ischemic heart disease (17, 19). Although the role of ANG II in various physiological and pathophysiological processes has been studied in numerous systems, assessment of its genome-wide influence began only recently (6, 7, 29, 73).

SAGE is a high-throughput method for quantitative evaluation of global gene expression (63). Similarly to microarray technology, SAGE can be used to evaluate genome-wide transcriptional response of a given cell, tissue, or organ to environmental stimuli. In contrast to microarrays, however, SAGE does not depend on a priori gene knowledge and thus represents a truly unbiased technique (53).

In this report we present results of differential gene expression profiling in the kidneys of mice after 7 days of continuous ANG II administration.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
The animals were housed in the animal quarters, and all experiments were conducted in accordance with the "Guidelines for the Care and Use of Animals" approved by the Boston University School of Medicine. The genetic background of mice used in this study is mainly C57BL/6J with a small contribution from 129/Sv and DBA/2J strains. Male littermates weighing 25–27 g were used in this study. Two mice received continuous ANG II infusion (0.9 µg/h) via subcutaneous osmotic minipumps (Alzet model 2001; Alza, Palo Alto, CA) for 7 days. Two control littermates were infused with saline. Minipumps were implanted under anesthesia with pentobarbital sodium (50 mg/kg ip), the animals were allowed an overnight recovery period, and the administration of ANG II or saline initiated the following day. Systolic BP and heart rate measurements were obtained daily in conscious mice using a computerized, noninvasive tail-cuff system (model BP 2000, Visitech Systems) as previously described (23). On day 7 of ANG II administration, the BP of infused mice was 156 ± 1 mmHg (mean ± SD), whereas the control littermates remained normotensive (106 ± 9 mmHg). After completion of BP measurements, mice were euthanized with CO2, and organs were harvested, placed in solution of RNAlater (Ambion, Austin, TX), and frozen at -80°C. The RNA prepared from the kidneys was used to construct control and hypertensive (ANG II-7dK) whole kidney SAGE libraries and for validation of results by quantitative real-time RT-PCR (Q-RT-PCR). To further verify differential expression of genes identified by SAGE, we conducted additional Q-RT-PCR analysis using total RNA prepared from the kidneys of a second group of animals, of different genetic background, that were administered ANG II in a separate experiment. C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, ME), and continuous infusion of either ANG II (n = 6) or saline (n = 6) was carried out for 7 days under the same protocol as described above. On day 7, BP of ANG II-infused and control animals in this group were, respectively, 156.5 ± 12.9 and 110.8 ± 9.5 mmHg.

Construction of SAGE libraries.
SAGE kidney libraries were generated essentially as described (63), with minor modifications. Total RNA was prepared using TRIzol reagent according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA). To avoid potential contamination with genomic DNA, total RNA preparation was treated with DNase (Invitrogen) in accordance with manufacturer’s protocol for DNase digestion and subsequent DNase removal (SNAP kit, Invitrogen). Poly(A) RNA was isolated from the total RNA with oligo-(dT)25-coated magnetic beads (Dynal, Oslo, Norway). Double-stranded cDNA synthesis (SuperScript II cDNA synthesis kit, Invitrogen), digestion with NlaIII (New England Biolabs, Beverly, MA), and ligation with custom-made (Integrated DNA Technologies) double-biotinylated 40-bp SAGE oligonucleotide DNA linkers 1 and 2 (63) has been carried out sequentially directly on the beads. After digestion with BsmFI (New England Biolabs) to release cDNA tags from the magnetic beads, the beads were pelleted, and released linker-tags were ethanol precipitated and blunted with T4-DNA polymerase (New England Biolabs). The L1- and L2-linker-tag complexes were then ligated together to form linker-"ditag"-linker constructs. A large-scale PCR amplification (4 x 96, 50-µl reactions) was carried out using biotinylated PCR primers, as previously described (43). Following amplification, PCR products were pooled, subjected to 12% preparative polyacrylamide gel electrophoresis (PAGE), and a 102-base pair DNA was excised, extracted using QIAEX II procedure (Qiagen, Hilden, Germany), and digested with NlaIII to release cDNA "ditag" sequences. Ditags were purified from biotinylated linkers by a two-step process, involving the use of streptavidin-coated magnetic beads (Dynal), followed by further purification through a 12% PAGE, and ligated to produce concatemers. The concatenated ditags were size-fractionated through an 8% PAGE, and gel regions between 300 and 800 bp were excised. To prevent contamination of concatemers with aggregates of ditags, the concatemerization reaction was heated for 15 min at 65°C prior to PAGE (25). Purified concatemers were cloned in the SphI site of pZero (Invitrogen). Following transformation of TOP10 E. coli electrocompetent cells (Invitrogen), Zeocin-resistant colonies were checked for the presence of an insert by direct PCR using M13 forward and reverse primers. PCR-amplified inserts of >=500 bp were sequenced with an automated sequencer (model ABI3700; PerkinElmer, Foster City, CA) using BigDye terminator cycle sequencing chemistry (PerkinElmer). All electropherograms were checked manually to resolve sequencing artifacts and ambiguous base calls.

SAGE data analysis.
Concatemer sequences of each clone were analyzed with the SAGE 2000 v. 4.12 software (provided by Dr. Kinzler’s laboratory at Johns Hopkins Cancer Center, Baltimore, MD) to identify individual tags. Tags corresponding to linker sequences were discarded, and duplicate dimers were counted only once. Tag identity was determined by sequence homology search of the murine SAGE database at the National Center for Biotechnology Information (NCBI). Comparison between the two SAGE libraries was carried out using statistical functions available in the SAGE 2000 software for P value calculations and Monte Carlo simulations, with a normalization value set to 11,300 tags per library and the minimal tag count setting of 2 for the two libraries combined. P < 0.05 was considered significant. Assignment of tags corresponding to differentially expressed genes was individually checked using SAGEmap (http://www.ncbi.nlm.nih.gov/sage/) and GGEG (Global Gene Expression Group; http://sciencepark.mdanderson.org/ggeg/search.htm) databases. Sequences of the corresponding differentially expressed mRNAs or expressed sequence tags (ESTs) were retrieved from the UniGene database and individually checked to verify that the tag corresponded to the most downstream NlaIII site.

Quantitative real-time RT-PCR.
The TaqMan reverse transcription (RT) reagents (Applied Biosystems) were used to synthesize oligo-(dT)16-primed cDNA in a 50-µl reaction containing 1 µg of DNase I-treated total RNA. RT reaction was carried out as suggested by the manufacturer at 25°C for 10 min, 48°C for 30 min, and 95°C for 5 min. The incubation step at 25°C is necessary for the RT reaction with oligo-(dT)16 primers to obtain the optimal results. The cDNA was analyzed immediately or stored at -20°C for later use. Q-RT-PCR reactions were performed with the ABI Prism 7900HT Sequence Detection System using a SYBR Green-based protocol (Applied Biosystems). Oligonucleotide primers for RT-PCR were designed with the Primer Express 2.0 software program (Applied Biosystems) and manufactured by Integrated DNA Technologies. All reactions were run in triplicates and included negative controls. The concentrations of the forward and reverse primers were between 300 and 500 nM. After initial denaturation at 95°C for 10 min, the cDNA products were amplified for 40 cycles consisting of denaturation at 95°C for 15 s, and annealing and extension was performed in a single step at 60°C for 1 min. The SDS 2.0 software generated standard curves from 10-fold serial cDNA dilutions, and the threshold cycle (Ct) was normalized for each standard curve. The range of slopes was between -2.63 and -3.70, where -3.33 corresponds to 100% efficiency of the PCR reaction. The copy numbers for all samples were normalized with the data obtained from GAPDH and/or ß-actin endogenous controls. Sequences of primer pairs used in Q-RT-PCR are shown in Table 1.


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Table 1. Primer sets used for Q-RT-PCR analysis

 

    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Following a 7-day continuous administration of ANG II, we constructed and compared whole kidney SAGE libraries from hypertensive (ANG II-7dK library) and control (control library) mice. As seen in Table 2, the number of SAGE tags analyzed was nearly equal for the two libraries. Sequencing of 11,320 tags from control and 11,677 tags from the ANG II-7dK libraries resulted, respectively, in an expression profile of 5,612 and 5,756 unique mRNA transcripts. Likewise, comparison of tags distribution between the two libraries revealed remarkable similarity in the overall expression profiles (Table 2). Approximately three quarters of the tags were observed only once in a given library, mimicking the expected low basic transcription level for the majority of mammalian genes (62). The remaining tags were detected two or more times, with a small fraction encountered 30 to several hundred times. The most abundant transcripts identified in control and ANG II-7dK libraries are listed in Tables 3 and 4, respectively, and the entire catalog of tags from both libraries is available at http://www.ncbi.nlm.nih.gov/sage/ (GEO accession numbers GSM9194 and GSM9195). The high levels of mitochondrial DNA-encoded transcripts are consistent with the high energy demand of the kidney and have been previously noted in kidney SAGE libraries by other investigators (16, 64). Likewise, most of the non-mitochondrial tags shown in Tables 3 and 4 have been previously detected at high frequency in whole kidney SAGE libraries (16, 64).


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Table 2. Summary of SAGE libraries

 

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Table 3. Highly expressed genes in the control SAGE library

 

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Table 4. Highly expressed genes in the ANG II-7dK SAGE library

 
Comparison of the ANG II-7dK and control libraries revealed a number of transcripts with significant (P < 0.05) difference in abundance, a subset of which is listed in Table 5.


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Table 5. Differentially expressed genes identified by SAGE

 
All differentially expressed genes identified by SAGE have been individually checked to confirm the tag-to-gene assignment. With two exceptions, all tags listed in Table 5 matched a gene or EST entry in current murine databases. This is not surprising, as the predominantly C57BL/6J genetic background of mice used in our study is the same for which the entire genome has been recently sequenced by the Mouse Genome Sequencing Consortium (68) and 60,770 full-length cDNAs annotated by the FANTOM Consortium and the RIKEN Genome Exploration Research Group (41). Two tags listed in Table 5 remain unassigned. One of these (tag TCCTATTAAG) has been previously detected in the whole kidney SAGE library prepared from ROP mice (16).

For all annotated genes, we assessed the position of the tag within the corresponding mRNA sequence. Only the genes with the tags derived from the NlaIII site nearest the poly(A) tail and/or polyadenylation signal have been selected for a subsequent follow-up. Likewise, the genes giving rise to more than one tag were not considered further. These criteria, however, have not been applied to the mitochondrially derived tags, as the mitochondrial transcripts are polycistronic and lack polyadenylation signals (58).

Given the modest number of tags sequenced and the resultant representation of many transcripts by a small number of tags, the differential expression of genes identified with SAGE was ascertained by the Q-RT-PCR method to eliminate false-positive findings. Only the nuclear genes with unambiguous assignment were chosen for additional analyses, whereas the mitochondrial transcripts and multiple matches (i.e., several genes that share the same tag) have not been evaluated. Of the 30 genes tested, differential expression was verified for 14 genes (Fig. 1A and Table 5). To ascertain the true influence of ANG II on the expression of these genes, we carried out the Q-RT-PCR analysis on the RNA from the kidneys of a second set of animals, of different genetic background, that were administered ANG II for 7 days in a separate experiment (as described in MATERIALS AND METHODS). Although statistical significance for some genes has not been reached, because of considerable interanimal variation, differential expression of five genes was confirmed (Fig. 1B).



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Fig. 1. A: validation of the differentially expressed genes identified with serial analysis of gene expression (SAGE) by quantitative real-time RT-PCR (Q-RT-PCR). Q-RT-PCR reactions, run in triplicates and including negative controls, were performed with SYBR Green using total RNA from the kidneys of mice used in SAGE library construction, as described in MATERIALS AND METHODS. The transcript copy numbers for all samples were normalized with the data obtained from Gapdh or ß-actin endogenous controls, and the average values were calculated. The y-axis shows the difference in the expression levels of genes (listed in alphabetical order on the x-axis) in the ANG II-7dK samples (n = 2) relative to the control (baseline) samples (n = 2), presented as (ANG II-7dK transcript copy number/Control transcript copy number) - 1. B: independent confirmation of the differential expression of 5 genes by Q-RT-PCR in a second group of mice. Q-RT-PCR reactions were performed with SYBR Green using total RNA from the kidneys of C57BL/6J mice, independently infused with ANG II or saline, as described in MATERIALS AND METHODS. The copy numbers for all samples were normalized with the data obtained from Gapdh endogenous control, and the means ± SE were calculated. Statistical analysis was performed by Student’s t-test, with the P value of <=0.05 considered statistically significant. The y-axis shows the difference in the expression levels of genes in the ANG II-7dK samples (n = 6) relative to the control (baseline) samples (n = 6), presented as (ANG II-7dK transcript copy number/Control mean transcript copy number) - 1.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Using SAGE, we identified a number of genes that are either up- or downregulated after a continuous 7-day ANG II administration, some of which, to our knowledge, have not been previously described in a context of angiotensin-mediated effects.

Remarkably, two genes [Klk1, encoding the tissue kallikrein (EC 3.4.21.35), and Ctsd, encoding cathepsin D (EC 3.4.23.5)], shown by SAGE and confirmed by Q-RT-PCR in two separate groups of mice of different genetic background to be upregulated in response to ANG II, map to chromosome 7 within the previously identified BP quantitative trait locus (Bpq7) (55). The Bpq7 has been mapped to a broad region, cM 20–60, with 95% confidence limits in the cM 35–50 interval (55). The map positions for Klk1 and Ctsd are, respectively, at 23 cM and 50 cM, making them likely candidate BP regulation genes. Whereas numerous studies in animal models (1, 9, 34, 44, 66, 67, 70) and in humans (72) provided ample evidence for the involvement of tissue kallikrein in BP regulation, the role of cathepsin D in HTN has not been assessed.

Cathepsin D, a lysosomal aspartic proteinase, has been extensively studied in a context of tumorigenesis (21), p53-dependent tumor suppression (71), apoptosis (14), cell growth and tissue homeostasis (49), and Alzheimer pathogenesis (4). Although the Ctsd gene is a known target of estrogen regulation (8), its regulation by ANG II, to our knowledge, has not been previously reported. Although the relationship between the ANG II-induced HTN and the upregulation of cathepsin D by ANG II remains to be explored, it may relate to the overactivation of the intrarenal renin-angiotensin system described in several experimental animal models of HTN (22, 65). Thus the upregulation of the ANG II precursor, angiotensinogen, by ANG II in the kidney (26, 51) suggested that a positive amplification mechanism involving a local renin-angiotensin system is, in part, responsible for the augmented intrarenal ANG II content in the animal models of HTN (39). Although transcription of the renin gene is known to be suppressed by ANG II (51), cathepsin D, colocalized with renin in the secretory granules of juxtaglomerular cells (59), can generate renin from the inactive prorenin precursor (38), as well as stimulate renin release (56). Thus upregulation of the cathepsin D detected by SAGE may be due to its involvement in a positive feedback loop of ANG II generation via the regulation of the granular renin stores available for secretion, necessary for the conversion of the angiotensinogen to angiotensin I in the kidney. Interestingly, stimulation of renin release by the renal kallikrein has also been reported (56, 57). Thus, although the observed upregulation of the Klk1 gene is likely due to its protective role in offsetting the hypertensive effects of the renin-angiotensin system, it may also be suggestive of its involvement in the intrarenal ANG II amplification cycle in this HTN model.

Recently, expression analysis utilizing microarray of a subset of rat (73) and human (6) genes identified changes in transcript profiles of genes associated with oxidative stress and pro-apoptotic pathways in the renal medulla of rats infused with subpressor doses of ANG II (73) and in in vitro cultures of human proximal tubular cells following a brief ANG II exposure (6). Indeed, ANG II has been shown to stimulate oxidative stress in several animal models of HTN (45), as well as to promote the oxidative stress-induced apoptosis in in vitro cultures of renal glomerular epithelial (15), mesangial (31), and proximal tubular (5) cells. Reduced levels of the mitochondrial ATPase subunit 6 transcripts in the ANG II-7dK library detected by SAGE in our study (Table 5) may be indicative of the oxidative stress-induced apoptosis (12, 20). In this context, increased expression of cathepsin D, an important mediator of the oxidative stress-induced apoptosis (24, 42, 46, 47), can be envisioned to mediate the ANG II-induced pro-apoptotic effects. In addition to its role in apoptosis, oxidative stress has been shown to cause vasoconstriction by several mechanisms, one of which is thought to operate by decreasing the basal level of nitric oxide (45). Interestingly, increase in tissue kallikrein expression has been shown in response to administration of a nitric oxide synthase inhibitor, L-NAME (9). Clearly, further investigation is needed to determine the putative role(s) of cathepsin D and renal kallikrein in ANG II-regulated pathways.

Downregulation of genes encoding metabolic enzymes (Hibadh, Temt, Hsd3b4) was detected by SAGE (Table 5). Interestingly, concordant downregulation was observed for the enzyme involved in androgen metabolism (Hsd3b4) and the kidney androgen-regulated protein (Kap) (Table 5). Genes for two transporter proteins, Vdac1 and Slc22a1l, are also downregulated (Table 5).

In addition to known genes, we identified several novel sequences regulated by ANG II (Table 5). Of these, two tags remain unassigned, as they do not match any gene in current databases and apparently represent yet unrecognized genes. One of these tags (TCCTATTAAG) has been previously detected in the whole kidney SAGE library prepared from ROP mice (16). In addition, two tags identified by SAGE correspond to cDNA sequences elucidated in a course of the mouse genome sequencing and annotation effort (41, 68) and are derived from genes with unknown function. BLAST (2) search of NCBI databases revealed the presence of genes with high sequence similarity in humans. Thus the 4833439L19Rik transcript, identified by virtue of its downregulation in ANG II-7dK library, maps to mouse chromosome 12 and encodes a putative protein of 303 amino acids, with 80% identity to an unnamed human protein predicted from a sequence on human chromosome 5q35.3. The HUGE ("Human Unidentified Gene-Encoded Large Proteins") database search revealed predominant expression of the gene in human brain, ovary, kidney, and liver. Another downregulated transcript, 0610012H03Rik, maps to mouse chromosome 2 and shares 86% identity with predicted protein encoded by a human gene on chromosome 11p13. The function of these novel genes and their putative regulation by ANG II remain to be elucidated.

We recognize that the number of differentially expressed genes presented in this report is likely to be an underestimate. First, 11,000 tags sequenced from each library allowed comparison between highly and moderately expressed genes, whereas genes expressed at a low level are not likely to be represented at this level of coverage. Likewise, tags corresponding to genes with expression limited to specific regions of the kidney may not be represented in sufficient number in the whole kidney SAGE library, which typically provides information on transcripts in the proximal tubular cells constituting the bulk of the kidney mass. It has been previously shown that transcriptional profiles of SAGE libraries generated from microdissected kidney tubules differ from that obtained from the whole kidney (64). Thus transcripts from genes encoding creatine kinase, uromodulin, and chloride channel CIC-K1 are enriched substantially in the SAGE library prepared from medullary thick ascending limbs of Henle’s loop, whereas tags matching aquaporin-2 and 11ß-hydroxysteroid dehydrogenase type 2 are much more abundant in the SAGE library made from the outer medullary collecting ducts (64). In this regard, it is of interest to note that tags derived from the renin gene have not been observed in the published mouse kidney SAGE libraries, and only a single tag was detected in the control SAGE library in our study. Since expression of the renin gene in the kidney is known to be localized to the juxtaglomerular cells lining the afferent arteriole (52), the expected transcriptional downregulation of renin by ANG II may not be evident from the analysis of a moderate number of tags.

In addition, there are known limitations of SAGE technology that bias the observed outcome of the quantitative SAGE evaluation. These include sequencing errors, nonuniqueness of tag sequences, differential RNA splicing, and DNA polymorphisms (54). Although rigorous analysis of SAGE tags employed in the present study likely minimized some confounding factors, such as sequencing errors, incorrect tag-to-gene assignments, and nonuniqueness of tag sequences, several biases remain. Thus genes devoid of NlaIII recognition site, an anchoring restriction enzyme used in SAGE library construction, as well as nonadenylated transcripts, are excluded from SAGE library and cannot be evaluated.

Although several statistical methods are now available for the quantitative comparison of SAGE libraries (3, 10, 27, 32, 35, 36, 60, 74), with the various tests differing to some degree in their specificity and sensitivity (33, 48), the optimal approach still remains controversial (50). Moreover, since each SAGE library provides only one measurement, the biological interanimal variation and the accuracy of experimentation cannot be assessed, which necessitates subsequent verification of results by an independent method. Thus a two-stage experimental design is warranted (37).

Since transcriptional profile has been obtained after 7 days of continuous ANG II administration, our present study does not discriminate between direct and downstream targets of ANG II. Moreover, differential expression detected by SAGE does not distinguish between transcriptional and posttranscriptional (i.e., message stability) mechanisms. Nor does it identify changes due to posttranslational events. Thus the SAGE method, similar to other widely used high-throughput technologies for transcriptional profiling, such as microarray and differential display, can provide only partial information in any given study. In contrast to other methodologies, however, the SAGE tag counts offer quantitative assessment of transcripts abundance and can be directly compared with libraries submitted to SAGE databases by other investigators.

In summary, using the SAGE method, we identified several genes previously not known to be regulated by ANG II. These genes provide probes for future studies of the physiological role of angiotensin and constitute useful models with which to explore the mechanisms of angiotensin-related pathologies.


    ACKNOWLEDGMENTS
 
We thank Dr. Kinzler for providing SAGE analysis software, and we thank Huai-Jen Yang, Kiran Thakur, and Liza Mermegas for superb technical assistance.

This work was supported by National Institutes of Health Grants P50-HL-55001 and U01-HL-66617.


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

Address for reprint requests and other correspondence: F. Schwartz, Dept. of Medicine, Hypertension Section, Boston Univ. School of Medicine, 700 Albany St., W508, Boston, MA 02118 (E-mail: fschw{at}bu.edu).

10.1152/physiolgenomics.00108.2003.


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 ABSTRACT
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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