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

Abstract

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.

Gene expression analysis of Tek/Tie2 signaling

“…Although the true identity of over one-half of these highly abundant tags could not be ascertained due to ambiguous mappings, a majority of these tags appeared to be involved in routine cellular processes such as energy metabolism and protein and lipid synthesis. This result is consistent with other SAGE libraries, reflecting the high demand for cell maintenance (6,41). In addition, a few of these highly abundant tags were mapped to hemoglobin and upregulated in the KO library; this indicates that Tie2 signaling may act to suppress erythropoiesis, or the elevated levels may represent the presence of red blood cells accumulating in the KO yolk sacs.…”

“…By performing Gene Ontology analysis to these NS tags after gene annotations, potential biological themes that were affected as a result of the experiment can be distinguished and selected for further studies. As observed in other SAGE analyses, genes implicated in metabolism and cellular growth are most abundant (6,41). This suggests that impaired signaling from the Tie2 receptor in KO yolk sacs may have a profound influence on normal cellular processes.…”

“…In fact, only ϳ0.2% of the tags were observed Ͼ100 times, while over two-thirds of the tags were singletons. Overall, the tag distribution profiles of the two SAGE libraries presented in this study are similar to other SAGE libraries, suggesting that the expression of the majority of mammalian genes is usually at a low level (6,41,45).…”

Novel targets of ANG II regulation in mouse heart identified by serial analysis of gene expression

“…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. Continuous infusion of 0.9 g/h ANG II (n ϭ 6) or saline (n ϭ 6) was carried out via subcutaneous osmotic minipumps (Alzet model 2001; Alza; Palo Alto, CA) for 7 days, as previously described (23,41). Systolic blood pressure (BP) and heart rate measurements were obtained daily in conscious mice using a computerized, noninvasive tail-cuff system (model BP 2000, Visitech Systems).…”

“…The TaqMan RT reagents (Applied Biosystems, Foster City, CA) were used to synthesize oligo-(dT) 16-primed cDNA in a 50-l reaction containing 1 g of DNase I-treated total RNA, as previously described (41). Q-RT-PCR was performed with the ABI Prism 7900HT Sequence Detection System using a SYBR greenbased protocol (Applied Biosystems).…”