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Am J Physiol Regul Integr Comp Physiol 284: R1219-R1230, 2003. First published January 23, 2003; doi:10.1152/ajpregu.00257.2002
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Vol. 284, Issue 5, R1219-R1230, May 2003

Gene expression reveals vulnerability to oxidative stress and interstitial fibrosis of renal outer medulla to nonhypertensive elevations of ANG II

Baozhi Yuan, Mingyu Liang, Zhizhang Yang, Elizabeth Rute, Norman Taylor, Michael Olivier, and Allen W. Cowley Jr.

Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study was designed to determine whether nonhypertensive elevations of plasma ANG II would modify the expression of genes involved in renal injury that could influence oxidative stress and extracellular matrix formation in the renal medulla using microarray, Northern, and Western blot techniques. Sprague-Dawley rats were infused intravenously with either ANG II (5 ng · kg-1 · min-1) or vehicle for 7 days (n = 6/group). Mean arterial pressure averaged 110 ± 0.6 mmHg during the control period and 113 ± 0.4 mmHg after ANG II. The mRNA of 1,751 genes (~80% of all currently known rat genes) that was differentially expressed (ANG II vs. saline) in renal outer and inner medulla was determined. The results of 12 hybridizations indicated that in response to ANG II, 11 genes were upregulated and 25 were downregulated in the outer medulla, while 11 were upregulated and 13 were downregulated in the inner medulla. These differentially expressed genes, most of which were not known previously to be affected by ANG II in the renal medulla, were found to group into eight physiological pathways known to influence renal injury and kidney function. Particularly, expression of several genes would be expected to increase oxidative stress and interstitial fibrosis in the outer medulla. Western blot analyses confirmed increased expression of transforming growth factor-beta 1 and collagen type IV proteins in the outer medulla. Results demonstrate that nonhypertensive elevations of plasma ANG II can significantly alter the expression of a variety of genes in the renal outer medulla and suggested the vulnerability of the renal outer medulla to the injurious effect of ANG II.

renal inner medulla; cDNA microarray


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE ROLE OF THE KIDNEY in the long-term regulation of arterial blood pressure is well recognized (4), and it is firmly established that the renin-angiotensin system is one of the most important hormonal pathways involved in the regulation of renal blood flow, glomerular filtration, and tubular function. It is evident that sufficient elevation of circulating ANG II resets the pressure-natriuresis relationship to higher operating levels, leading to renal sodium retention and hypertension (4, 6).

Most studies evaluating the effects of ANG II, however, have been directed either at whole kidney function or the function of renal cortical structures. The effects of ANG II on the function of the renal medulla have remained relatively obscure despite numerous studies having now established that medullary blood flow and tubular function can importantly influence sodium excretion and long-term blood pressure regulation (4-6). There are also data indicating that the renal medulla is protected in large measure from the vasoconstrictor actions of ANG II due to stimulation and release of large amounts of nitric oxide (40, 49). This could be further buffered by ANG II-induced increases of prostaglandin E (31, 41) and kinins (23, 42). It has also been observed that ANG II administered intravenously in low concentrations significantly reduces renal medullary blood flow only when medullary production of nitric oxide is inhibited (45).

It is now well established that ANG II is a renal growth factor that modulates cell growth and extracellular matrix synthesis and degradation (10, 37, 47). However, until this time, studies have utilized doses of ANG II that exceed those seen even under extreme pathophysiological conditions, and in studies in which this peptide has been administered chronically into experimental animals for several weeks, it has been difficult to ascertain whether changes in kidney function and end-organ pathology were a result of the high levels of renal perfusion pressure associated with hypertension or direct effects of high levels of circulating ANG II.

To reveal the diversified physiological influences of small sustained elevations of ANG II in renal medulla, cDNA microarray studies were designed to identify pathways that could be changed in response to ANG II stimulation. We hypothesized that even nonhypertensive elevations of plasma ANG II would modify the expression of genes involved in renal injury that could influence oxidative stress, extracellular matrix formation, and functional aspects of the renal medulla. Toward this end, a nonhypertensive dose of ANG II was utilized that has been shown to raise plasma ANG II from 11.3 to 19.7 pg/ml in the absence of chronic elevations of mean arterial pressure (MAP; Refs. 27, 45, 49).

The application of cDNA microarray techniques enabled us to examine changes in the expression of thousands of genes at one time. Although it is now possible to stamp nearly 20,000 cDNAs or expressed sequence tags (ESTs) of many genes on a single glass microscope slide (17), at this time the specific genes related to these ESTs have been annotated for only ~2,000 rat genes. Furthermore, the cost of amplifying and stamping custom arrays or purchasing large arrays (~20-30,000) is considerable. It is also difficult to relate the expression of unknown ESTs to physiological significance. The analysis and interpretation of such large datasets are also problematic. For this reason, in the present study we chose to evaluate the effects of ANG II on the expression of genes within the renal medulla using a relatively small and more affordable microarray containing 1,751 rat genes, which include nearly 80% of all rat genes annotated with some known function in some tissue. This set of cDNAs was recently used to study gene expressions in the renal medulla of Dahl salt-sensitive rats (20, 21). The other important aspect of the present study was related to the replication of data based on results obtained from the kidneys of six individual rats chronically infused with ANG II and compared with six vehicle-infused rats.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Chronic intravenous infusion of ANG II and blood pressure measurements. Experiments were performed on adult male Sprague-Dawley rats (Harlan, Madison, WI). The rats were housed in the Animal Resource Center at the Medical College of Wisconsin, with food and water provided ad libitum throughout the experimental protocol. All procedures were approved by the Medical College of Wisconsin Animal Care Committee and followed the guiding principles of Institutional Laboratory Animal Resources Guide for Care and Use of Laboratory Animals.

At 11 wk of age, the rats were anesthetized with ketamine (10 mg/100 g) and acepromazine (0.5 mg/100 g). The femoral artery and vein were catheterized as described previously (7) for the chronic measurement of blood pressure and intravenous infusion. The animals were allowed 7 days to recover from the surgery. Rats were maintained on a normal sodium diet throughout the study (~1% NaCl, Purina Rat Chow). Arterial pressure was then measured for 3 h daily (9:00- 12:00 PM) for 5 control days while receiving an intravenous infusion (6 ml/day) of 0.9% NaCl solution. In six rats, the solution was then switched to a subpressor dose of ANG II (5 ng · kg-1 · min-1) that was infused intravenously continuously for 7 days during which time arterial pressure was measured daily. Another six rats continued to receive normal saline and served as vehicle controls.

Construction of rat known gene cDNA microarrays. Microarrays of 1,751 cDNA clones were constructed as described previously (20) using 1,687 rat gene cDNA clones purchased from Research Genetics (Huntsville) and 64 rat genes cloned previously in our department. Each clone was amplified by PCR using T7/T3 universal primers. Each PCR product was analyzed by agarose gel electrophoresis. Approximately 90% of the PCR products appeared as single bands. The PCR products were then diluted in 50% DMSO and spotted in duplicate on glass microscope slides (Corning Glass Works, Corning, NY) coated with poly-L-lysine using a four-pin arrayer (Affymetrix, Santa Clara, CA). Several negative controls such as PCR buffer with primers and empty vectors were spotted at 372 various places on the slide. Housekeeping genes such as beta -actin and GAPDH PCR products were diluted in a series dilution and then spotted in various places on the slide. Slides were blocked after the procedure described previously (20) and stored at room temperature in the dark.

Tissue preparation, cDNA labeling, and microarray hybridization. On completion of ANG II or saline infusion, rats were anesthetized, and kidneys were removed as described previously (48). Renal inner medulla and outer medulla were selectively dissected and snap-frozen. Total RNA was isolated from these tissues using TRIzol Reagent (Life Technologies, NY) following the manufacturer's protocol. The quality of the RNA was determined by a total absorbance scan from 200- to 450-nm wavelength to ensure that the absorbance ratio of 260/280 was >= 1.8. The absorbance reading of the RNA was required to give a smooth peak at ~260-nm wavelength, indicating elimination of phenol contamination from extractions. The total RNA samples from the kidneys of the control and ANG II-infused rats were randomly paired in labeling and hybridization. Two micrograms of total RNA from each sample was reverse-transcribed and labeled with either fluorescein- or biotin-dCTP using TSA Labeling and Detection Kit (MICROMAX, NEN Life Science Products, Boston, MA). The two cDNA samples (control and ANG II infused) were then pooled and hybridized to one microarray slide at 65°C overnight. The slide was then washed three times at 5 min each in 0.5× standard saline citrate (SSC), 0.01% SDS; 0.06× SSC, 0.01% SDS; and 0.06× SSC solution, respectively. After washing, the slide was incubated with anti-fluorescein conjugated with horseradish peroxidase (HRP), which catalyzed the deposition of Cyanine 3 (Cy3)-labeled tyramide reagent. HRP was then inactivated before the second streptavidin-HRP application, which catalyzed the deposition of Cyanine 5 (Cy5)-labeled tyramide reagent. Slides were washed once with 0.06× SSC solution and scanned using the ScanArray 5000 (Packard Bioscience, Meriden, CT) to quantify the fluorescent intensity of Cy3 and Cy5 in each spot.

Microarray data analysis. The fluorescent intensities from both Cy3 and Cy5 were extracted from the microarray images using ImaGene 4.20 software (BioDiscovery, Los Angeles, CA) and analyzed as described previously with modifications (20, 21). Negative controls such as PCR buffer mix, clone vectors, and DMSO solution were included in each microarray. A total of 372 spots including replicates were pooled, and the mean and SD of their signal intensity were calculated for both Cy3 and Cy5. A spot was defined as "nondetectable" if its mean signal intensity was less than the mean of the negative control signal intensities plus two times the negative control SD for the same slide. A spot was defined as "low quality" if its mean signal intensity was less than its local background mean intensity plus two times the local background SD. If the spots were not designated as low quality or nondetectable, they were used in the analysis. The resulting dataset, designated as the defined raw data, underwent further analysis using Student's t-test after normalization with the intensities from Cy3 and Cy5 channels of the housekeeping gene beta -actin. Specifically, normalization was carried out using the average intensities of 310 beta -actin spots that were stamped on each slide. After the initial data selection as described above, an average of 290 beta -actin spots was utilized from each of the six slides hybridized from tissue obtained from the total RNA of both the renal outer medulla and inner medulla. The individual intensities of all six slides were averaged. A ratio was then calculated between the average overall intensity of the six slides and the average intensity of each slide. This ratio was used to correct the individual intensity of each hybridized slide. Student's t-test was performed for each individual gene by comparing the intensities from saline-infused samples with the ANG II-infused samples using the normalized intensities from both channels. A gene was considered differentially expressed significantly if its P value was <= 0.05.

Validation of microarray results with Northern blot analysis and resequencing cDNA clones. Eighteen and 13 genes were randomly selected from outer medulla and inner medulla, respectively, for Northern blot analysis. Twenty and 15 µg of total RNA from outer and inner medulla, respectively, were denatured and electrophoresed on 1.5% agarose gel containing 10% formaldehyde. The RNA was transferred onto positively charged nylon membrane and UV-crosslinked as described previously (48). The specific probes were generated by PCR using the specific clones as the templates. The blot was hybridized following the protocol described previously (48). After hybridization, the probe was stripped by boiling with 5% SDS solution. The blot was then hybridized using another probe. This protocol enabled us to use the same RNA membrane to hybridize with five different specific probes. The data were expressed as the ratio of the intensities of ANG II-infused total RNA to saline-infused total RNA.

After the differentially expressed genes were identified from the microarray analysis, the corresponding original cDNA clones obtained from Research Genetics were amplified with PCR. The PCR products were then purified and sequenced using BDT chemistry (Applied Biosystems, Foster City, CA) to verify their identity. Sequence homologies were identified using the BLAST search algorithm. Those clones that did not sequence on this first pass and were of greatest interest were sent to Seqwright (Houston, TX) for sequencing, and the sequence homologies were identified in the same way as the initial clone sequences.

Western blot analysis of transforming growth factor-beta 1 and collagen type IV protein levels in the outer medulla. Another two groups of rats were catheterized, and ANG II (5 ng · kg-1 · min-1) was infused for 7 days following the protocol described above. After 7 days of either ANG II (n = 6 rats) or saline infusion (n = 6 rats), the rats were anesthetized, and the kidneys were dissected. The renal outer medulla was then isolated, and protein extracts were prepared using a modified protocol described previously (19). Briefly, tissues were homogenized in sucrose buffer containing 20 mM HEPES, 1 mM EDTA, 255 mM sucrose, 0.4 mM phenylmethylsulfonyl fluoride, and 2 µg/ml leupeptin (all obtained from Sigma). The homogenate was then centrifuged at 1,500 g for 15 min, and the supernatant containing the protein was then aliquoted and stored at -80°C until use.

An aliquot of 200 µg of protein extract was electrophoresed in 12% SDS-PAGE gel and transferred onto a nitrocellulose membrane. The membrane was then washed and probed with 1:500 specific anti-rat transforming growth factor (TGF)-beta 1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and subsequently with 1:4,000 goat anti-rabbit IgG conjugated with HRP. This antibody recognized a specific protein with a size of 12.5 kDa corresponding to TGF-beta 1. To detect the immunoblotting signal, 8 ml of chemiluminescence detection solution (ECL solution, Pierce, Rockford, IL) was added, and the membrane was wrapped and exposed to Kodak film. The antibody was then stripped off by incubation in reprobing solution (62.5 mM Tris · HCl, 2% SDS, and 100 mM 2-mercaptoethanol, pH 6.7) for 30 min, 50°C. The membrane was then blocked and probed with 1:50 specific monoclonal mouse anti-human collagen type IV antibody (DakoCytomation, Carpinteria, CA), which recognized a band at 105 kDa corresponding to collagen type IV. This antibody shows a distinct cross-reactivity with bovine and rat collagen IV. The membrane was subsequently incubated with 1:3,000 goat anti-mouse IgG conjugated with HRP. After the chemiluminescence detection, the membrane was stripped again and probed with specific anti-beta -actin antibody (42 kDa) to verify the loading equivalence among samples.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of chronic infusion of ANG II at a subpressor dose on blood pressure. The MAP did not change significantly in either saline- or ANG II-infused (5 ng · kg-1 · min-1) rats (Fig. 1). MAP averaged 110 ± 0.6 mmHg during control period and 113 ± 0.4 mmHg in rats receiving ANG II. In saline-infused rats, MAP averaged 111 ± 0.3 mmHg during the control period and 109 ± 0.5 mmHg during the 7-day experimental period corresponding to the ANG II infusion.


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  		Fig. 1.   Effects of a subpressor dose of ANG II (5 ng · kg-1 · day-1; closed symbols) or saline (open symbols) on mean arterial blood pressure (MAP) in Sprague-Dawley rats. Blood pressure was measured daily for 3 h from indwelling arterial catheters.

Changes in mRNA expression profiles in the renal outer medulla. Total RNAs from six ANG II- and six saline-infused rats were randomly paired for hybridization on microarray slides (Fig. 2). To ensure equal labeling of the two dyes, RNAs from three pairs of rats were labeled with the two dyes switched between saline- and ANG II-infused rats. As the result of six hybridizations, 603 genes (34% of total genes) in the outer medulla passed the data selection process and yielded ratios from six hybridizations that were used for identification of differential expressions. Table 1 lists the differentially expressed genes in the outer medulla in response to ANG II infusion and categorizes them into eight potentially important mechanistic pathways in which they might be involved. A total of 36 genes were differentially expressed in the outer medulla in response to small elevations of plasma ANG II. Among them, 25 genes were downregulated, and 12 genes were upregulated based on the Student's t-test statistical analysis. Figure 3 summarizes the association of four of these pathways, based on the limited annotation available for these genes, and as hypothesized for their relationship to renal injury, extracellular matrix restructuring, and fluid and electrolyte homeostasis. As we had hypothesized, a number of these genes (n = 18) have been associated with oxidative stress, fibrosis, cell growth, and apoptosis. The differentially expressed genes related to oxidative stress responses in the outer medulla included copper-zinc-containing superoxide dismutase, D-dopachrome tautomerase, glutathione-S-transferase, metallothionein, NAD+-isocitrate dehydrogenase, and platelet-activating factor acetylhydrolase alpha 1, all of which were downregulated. Differentially expressed genes involved in fibrosis and cell apoptosis pathways were also identified. Specifically, cathepsin K, Tamm-Horsfall protein gene, thymosin beta-4 gene, tissue inhibitor of metalloproteinase-1, tissue-nonspecific alkaline phosphatase, and dipeptidyl peptidase 4 were all downregulated by ANG II.


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  		Fig. 2.   A false-color microarray image from renal outer medullary tissue demonstrating the cDNA microarray design. A total of 1,751 genes were spotted in duplicate on the slide using a 4-pin arrayer. Several negative control spots including DMSO solution, PCR buffer with and without primers, and clone vectors were also printed on the slide to serve as controls for data analysis. Signal and local background intensities were defined by ImaGene 4.20 software.


                              
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  		Table 1.   Significantly differentially expressed genes in renal outer medulla in response to intravenous ANG II



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  		Fig. 3.   Analysis of microarray results indicated that genes were differentially expressed between ANG II-infused and saline-infused animals in both the inner and outer renal medulla. The 36 genes differentially expressed in the outer medulla (see Table 1) and the 24 genes differentially expressed in the inner medulla (see Table 2) were grouped into 8 pathways of physiological importance in blood pressure and the production of renal injury. Four of these pathways are depicted in this schematic to suggest the relationships between these pathways and renal injury and fluid and electrolyte homeostasis. It is important to recognize that at this low dose of ANG II, there are no functional changes observed in blood pressure (Fig. 1). The changes in gene expression measured at this low dose suggest early changes in these pathways in the renal medulla that would lead to renal injury and hypertension.

Other differentially expressed genes were also identified. These included genes that are known to participate in sodium transport and reabsorption, pressor actions, metabolism/toxicity, and nuclear protein and transcriptional factor pathways. For example, several differentially expressed genes are known to be responsible for sodium transport and reabsorption such as a bumetamide-sensitive sodium-potassium chloride cotransporter and Na-K-ATPase alpha 1 and beta 3, which were upregulated by ANG II stimulation. Furthermore, the gene for the amiloride-sensitive Na+ channel was downregulated by ANG II in the outer medulla. These and other genes that could alter either blood flow or tubular transport in the outer medulla were influenced by ANG II. Medullary vascular tone may have been influenced by changes in expression of genes such as epoxide hydrolase 1, cytochrome P-450 4A8 enzyme, and inositol 1,4,5-trisphosphate receptor type 1, which were downregulated, and the cyclophilin gene, thought to be involved in hypertension in spontaneously hypertensive rats (16), which was upregulated.

Northern blot analyses were performed using mRNA extracted from the outer medulla for 18 randomly selected genes to determine differential expression. Expression ratios obtained from the microarray analysis (see Table 3) were highly consistent with those from Northern blot analysis. The log of these ratios exhibited a correlation coefficient equal to 0.855 (P < 0.0001) between the two methods.

Changes in mRNA expression profiles in the renal inner medulla. Six hundred forty two genes (37% of total genes) in the inner medulla passed the selection process and were analyzed for the expression differences. A total of 24 genes were differentially expressed in the inner medulla in response to small elevations of ANG II. Specifically, 11 genes were upregulated and 13 were downregulated in response to ANG II (Table 2). Again, these differentially expressed genes were found to be generally related to the physiological pathways similar to those in the outer medulla. Specifically, ANG II resulted in the differential expression of several genes that appear to be involved in formation and degradation of the extracellular matrix. Among these were cathepsin B, matrix gla protein, and cystatin beta. Cathepsin B gene was upregulated and cystatin beta gene was downregulated; both were thought to be involved in extracellular matrix breakdown. Genes associated with cell growth and proliferation were differentially expressed, including cofilin, insulin-like growth factor-binding protein 5, and nucleolin gene. Others related to oxidative stress were upregulated, such as aldehyde reductase I and betaine-homocysteine methyl transferase (BHMT). Some genes involved in the apoptotic processes, such as growth arrest- and DNA damage-inducible gene 153 (GADD153), were downregulated; others such as prosaposin (sulfated glycoprotein, sphingolipid hydrolase activator) gene were upregulated.

                              
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  		Table 2.   Significantly differentially expressed genes in renal inner medulla in response to intravenous ANG II

Other genes associated with various tubular transporters and signal transduction were also changed by ANG II stimulation. Of particular interest to our own laboratory, the bumetamide-sensitive sodium-potassium chloride cotransporter and Na-K-ATPase genes were both upregulated in response to the small elevations of ANG II, indicating that sodium transport and reabsorption may be enhanced in renal inner medulla.

The results were also verified by Northern blot analysis using 13 randomly selected genes. Again, a high level of consistency was found between the array and Northern blot (Table 3), yielding a correlation coefficient of 0.855 (P < 0.0001) based on log ratios of ANG II-infused animals vs. saline-infused controls.

                              
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  		Table 3.   Comparison of Northern blot and microarray methods for gene expression

Resequencing of cDNA clones. The original cDNA clones showing differential expression in microarray hybridizations in response to ANG II were examined by resequencing. Among 58 genes that differentially expressed in renal medulla (36 genes in the outer medulla; 24 in the inner medulla; with 2 of the same genes expressed in both the inner and outer), 49 have been sequence verified and are indicated with an asterisk after the gene name in Tables 1 and 2. Of the 50 clones sequenced, one did not match the identity given by Research Genetics.

Effects of intravenous ANG II (5 ng · kg-1 · min-1) on TGF-beta 1 and collagen type IV protein expression in renal outer medulla. Western blot analysis revealed that chronic administration of a subpressor dose of ANG II caused a significant increase in TGF-beta 1 and collagen type IV protein in the outer medulla compared with the outer medulla of saline-infused rats. As shown in Fig. 4, after 7 days of ANG II infusion, the normalized ratio of TGF-beta 1 protein was increased 25.9% over the response seen with saline infusion (1.259 ± 0.088 vs. 0.988 ± 0.077, respectively). The collagen type IV protein was increased 18.3% compared with the response of the saline-infused controls (1.183 ± 0.048 vs. 1.00 ± 0.060, respectively).


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  		Fig. 4.   Protein from renal outer medulla was probed for the presence of collagen type IV and transforming growth factor (TGF)-beta . Results from Western blot analysis normalized for beta -actin are summarized. Both proteins were stimulated by this low dose of ANG II. * P < 0.05, saline compared with ANG II infused (n = 6/group).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Microarray technology provides a powerful tool for physiologists to study the interplay of signals and transcriptional responses in complex biological systems. Although ANG II has been studied intensively for more than 40 years and is known to be of great physiological importance, the methods available until this time have restricted the measurement of RNA to only one or several elements of the signaling pathways. The present study enabled the screening of nearly 80% of all rat genes that have been identified at this time (i.e., DNA known to transcribe a known protein). The known genes were chosen for this analysis for two reasons. First, in many cases there is knowledge of the function(s) of the expressed proteins of these genes and varying degrees of understanding of the biological pathways into which they fit. Second, in our effort to custom design a chip that could be affordably used for the desired replication studies, it made sense to choose those rat genes about which we currently had the greatest knowledge.

Emphasis in the present study was placed on evaluating the effects of elevations of ANG II that occur under physiological conditions, such as a low-salt diet (27). Genes that were differentially expressed within the renal medulla were of special interest because it has been shown that changes in medullary blood flow or tubular reabsorption can importantly influence sodium and water homeostasis and chronic levels of arterial pressure (5). Although renin production occurs exclusively in the cortical juxtaglomerular cells, studies have shown that circulating ANG II, as well as the locally produced renal ANG II (29), may act acutely on the renal medullary vasa recta vessels (26, 33) and tubules of this kidney region. There is little known, however, about the chronic effects of small elevations of ANG II on the structure and function of the renal medulla of the adult kidney. This region was divided into outer (red) and inner (white) medulla and analyzed separately in the present study. The outer medulla is generally understood to be an important site of sodium reabsorption (medullary thick ascending limb), and the inner medullary collecting duct participates importantly in urine-concentrating mechanisms and volume regulation.

Evidence for activation of pathways that enhance oxidative stress and modify medullary extracellular matrix. The results support the hypothesis that sustained nonhypertensive elevations of circulating ANG II results in activation of pathways that increase oxidative stress and modify the extracellular matrix of the renal medulla. Johnson et al. (15) reported that hypertension produced by chronic administration of very large doses of ANG II (~ 400 ng · kg-1 · min-1) for 14 days induced marked vascular, glomerular, and tubulointerstitial injury with cell proliferation. There was increased expression of smooth muscle actin by mesangial cells and desmin by visceral glomerular epithelial cells. Most relevant to the present study, the kidneys of these Sprague-Dawley rats developed focal tubulointerstitial injury as well as tubular atrophy and dilation. Cast formation and interstitial monocytic infiltrate and mild interstitial fibrosis with increased type IV collagen deposition were observed. As recently reviewed by Mezzano et al. (24), some of these effects are mediated by growth factors, such as TGF-beta . Other effects appear to be due to ANG II acting as a proinflammatory cytokine, participating in various steps of the inflammatory process. Taken together, these studies have shown that high, nonphysiological elevations of ANG II can activate mononuclear cells and increase proinflammatory mediators, such as cytokines, chemokines, adhesion molecules, and NF-kappa B, and influence matrix degradation.

In all of these studies, because hypertensive doses of ANG II were administered, it must be recognized that it was unclear whether the observed end-organ damage resulted from the sustained elevation of arterial pressure or from the direct effects of ANG II. It has been shown that ANG II in high concentrations can stimulate oxidative stress and contribute to the vascular alterations associated with its hypertensive effects (2). The results of the present study demonstrate that even small, nonhypertensive elevations of circulating ANG II can increase levels of oxidative stress within the renal medulla and initiate changes in genes known to be involved in pathways responsible for renal fibrosis. This indicates that such responses are relevant to the normal control of renal function. Interestingly, ANG II at the subpressor dose used in the present study decreased the expression of glutathione-S-transferase mu type, metallothionein, and copper-zinc-containing superoxide dismutase (SOD) in the outer medulla (Table 1). These genes are generally recognized to be involved in protection against cellular stress so the suppression of these genes by ANG II might promote cellular oxidative stress by compromising these protective mechanisms. Furthermore, the gene of NAD+-isocitrate dehydrogenase was also decreased (18). NAD+-isocitrate dehydrogenase is critically important in cellular defense against oxidative damage by producing NADPH, which is important for the regeneration of reduced glutathione. Because NADPH is an important cofactor in many biosynthetic pathways, cells with low levels of NAD+-isocitrate dehydrogenase become more sensitive to oxidative damage when exposed to increasing amounts of H2O2 or menadione (14), and a reduction in the expression of this enzyme would be expected to result in a pro-oxidant state (18).

It is interesting that differences in the gene expression of aldehyde reductase I and BHMT in response to ANG II were observed between the outer and inner medulla (Tables 1 and 2). Upregulation of these genes was found in the renal inner medulla, but not in the outer medulla. Aldehyde reductase is a member of the aldo-keto reductase superfamily and metabolizes toxic aldehydes generated by lipid peroxidation (30). The enzyme is found in rat kidney (32), and we have shown in a previous study that Dahl S rats exhibit renal medullary upregulation of this gene in response to a high-salt diet (20). Similarly, upregulation of this enzyme has been found in the heart where it was found to reduce the generation of lipid peroxidation products associated with ischemia-reperfusion injury (39). BHMT, which was similarly upregulated, is a methyltransferase that catalyzes the conversion of homocystein to methionine using betaine as a methyl donor (11). Methionine residues that reside intracellularly may serve as antioxidants to scavage reactive oxygen species (43). So, with the upregulation of aldehyde reductase and BHMT occurring only in the inner medulla in response to ANG II, the results of the present study suggest that the cell defense system to oxidative stress may be stronger in the inner medulla compared with the outer medulla.

Other ANG II-induced responses were associated with a reduced protection of the outer medulla from the effects of oxidative stress. ANG II resulted in the differential expression of a number genes related to the formation of extracellular matrix and fibrosis (Table 1). For example, in the outer medulla, ANG II caused the decrease in cathepsin K and dipeptidyl peptidase IV (DPPIV) mRNAs. Cathepsin K is a cysteine proteinase that plays an important role in degradation of type 1 collagen. DPPIV is an enzyme that enhances the release of dipeptides from polypeptides to degrade collagen. A reduction in the expression of these two enzymes in the outer medulla suggests that the small elevations of ANG II in the present study were stimulating the formation of extracellular matrix and leading to fibrosis in outer medullary region. In contrast, in the inner medulla, an increase in cathepsin B expression and decrease in cystatin beta expression suggest once again that the inner medulla would be better protected from fibrosis in that these changes would increase the breakdown of collagen protein, which is the major accumulated protein related to tubular fibrosis.

A number of genes related to cell growth or programmed death were differentially expressed in rats administered ANG II in the present study. In the outer medulla, these included the upregulation of thymosin beta-4, CD24 antigen, and vascular endothelial growth factor (VEGF). Conversely, downregulation was observed of ornithine decarboxylase, DPPIV, cyclin D2, insulin-like growth factor 1, and perchloric acid-soluble protein. Although the functional annotation available for genes varies considerably and virtually no information exists specifically for cell types of the renal medulla, evidence in other tissues indicates that upregulation of the thymosin beta-4 gene increases collagen deposition, angiogenesis, wound healing, and remodeling of neuronal processes (28). Similar effects on cell growth have been found with insulin-like growth factor-binding protein 5 (25) and VEGF gene expression, which has been well characterized in the kidney (36).

Studies have demonstrated that ANG II induces apoptosis in renal cortical glomerular epithelial cells (9), but such responses have not been previously observed within the renal medulla. ANG II-induced differential expression of other genes in this study would also be expected to lead to apoptotic events within the renal medulla. The CD24 gene was found to be upregulated in renal outer medulla in the present study. Also referred to as heat-stable Ag (HAS), this gene has been found to be involved in apoptosis (44). In contrast, cyclin D2, an important regulator of the cell cycle that stimulates cell growth (44), was downregulated in the outer medulla. These expression changes suggest that these genes could be associated with the initial stages of fibrotic events in the outer medulla, apoptotic events that would be consistent with those events found to occur in the pathogenesis and progression of cardiovascular disease (8, 13).

Conversely, in the inner medulla, GADD 153 mRNA was downregulated (Tables 1 and 2), an event that would be expected to reduce apoptosis and protect against the apoptotic effects of ANG II. There is evidence that activation of the peroxisome proliferator-activated recepter (PPAR)-gamma may cause growth inhibition and apoptosis through GADD153 gene expression (38), so the downregulation of this gene would be expected to counteract apoptosis. Consistent with these changes, prosaposin was upregulated in the inner medulla. Prosaposin is the precursor of a sphingolipid activator protein and could also have a protective effect against apoptosis. It has been shown to be expressed at high levels in cerebellum during cell proliferation and maturation (46). Insulin-like growth factor-binding protein 5 was also upregulated, which would also be expected to have a protective effect against apoptosis (25).

Direct evidence for increased matrix protein in the outer medulla by Western blot analysis. To examine whether the ANG II-induced differential expression of genes within these pathways may indeed be leading to changes in protein indexes of medullary fibrosis, two additional groups of rats were studied to obtain sufficient medullary tissue to carry out Western blot analysis of TGF-beta 1 and collagen type IV proteins. Both proteins have been shown to be increased in tissues that have undergone fibrosis. The results of this study indicated that nonhypertensive elevations of ANG II can lead to detectable changes in the structural proteins of the renal medulla. However, the mRNA of these two genes was not significantly changed in the present analysis, suggesting posttranscriptional modifications that may have increased the expression of these proteins. Detailed studies will be required to identify the specific mediators of these changes and to localize the cells responsible for the release of the growth factors related to such changes.

ANG II effects on ion transporter pathways in the renal medulla. The results of the present study revealed a number of unexpected and novel pathways whereby ANG II may alter the expression of genes and tubular transport function within the renal medulla. Although expression differences do not ensure that changes also occurred in the steady-state expression of the associated enzymes or proteins, when multiple genes within a functional pathway are changed together it is more likely that these pathways were indeed modified.

A particularly interesting observation (Tables 1 and 2) was that ANG II resulted in the differential expression of genes involved in sodium reabsorption within the renal medulla. Effects of ANG II on sodium reabsorption in the renal cortex have been known since the late 1970s; however, it has remained unclear and controversial (1, 22) whether ANG II can influence Na+ reabsorption in the medullary thick ascending limb of the loop of Henle. This nephron segment, which is responsible for the reabsorption of 20-25% of the filtered sodium load, plays a key role in sodium and water homeostasis (12). As seen in Tables 1 and 2, ANG II upregulated the Na-K-2Cl (NKCC2) transporter mRNA and Na-K-ATPase alpha 1 mRNA in both the outer and inner medulla. Na+-K+-ATPase beta 3 subunit was also upregulated, but only in the outer medulla. Conversely, the amiloride-sensitive Na+ channel (eNaC) mRNA was downregulated in the outer medulla. Although previous studies have shown that ANG II directly stimulates eNaC activity in the cortical collecting duct (35), the present results indicate opposite effects on eNaC mRNA in the renal medulla. These observations suggest a number of followup studies that should be carried out to determine the functional relevance of these responses. Given the localization of the mRNA for Na+ channels in the medulla, the data indicate that subpressor elevations of ANG II may increase the transport mechanisms for Na+ reabsorption in the thick ascending limb by increasing apical sodium entry (through NKCC2) and basolateral extrusion of sodium (through Na+-K+-ATPase), while reducing Na+ reabsorption in the collecting ducts of the outer medulla (through eNaC).

Cysteine deoxygenase (CDO) is another gene that was differentially expressed in the outer medulla that may participate indirectly in sodium reabsorption. It was downregulated in response to ANG II stimulation. CDO catalyzes the conversion of cysteine to cysteine sulfinic acid and controls the rate-limiting step of sulfate production, which is an osmolyte in the renal medulla (34). It is also involved in the biosynthesis of the osmolyte taurine from cysteine through the so-called cysteine sulfinate pathway (3). The downregulation of CDO in combination with the upregulation of Na+ transporters would suggest that small elevations of ANG II may have reduced intracellular osmolyte content while increasing tubular sodium reabsorption.

Analytical assessment of the microarray data. A conservative yet robust analytical approach was used in the present study to analyze the dataset. The defined raw data as described in METHODS represented the basic dataset used to calculate the actual ratio for each gene in the experiment, thereby eliminating low-intensity and poor-quality signals. Importantly, microarrays were carried out in tissues obtained from six replicate animals, and, in addition, genes were stamped in duplicate on each array. In any physiological microarray study there are inherent variations between experimental animals and between stamped microarrays. Replicate studies are essential to establish the statistical significance, and in the present study the differentially expressed genes were identified using six replicates of each group and by applying the Student's t-test. These analyses were carried out only after normalization using the average intensities of beta -actin in both channels, a necessary step when trying to make comparisons between multiple hybridizations because variations in experimental conditions clearly contribute to quantitative differences observed between samples. Data-analysis techniques for microarray data continue to evolve. In our previous studies (20, 21), data were normalized by adjusting the mean ln(ratio) of Cy3 and Cy5 signals to be zero, based on the assumption that the expression levels of the vast majority of genes did not differ between the two samples being compared. In the present study, we used the expression of beta -actin, the housekeeping gene, as the normalization factor. This method also assumes similarities about the behavior of analyzed parameters and assumes that the distribution of genes is normal or unchanged by the ANG II. If the expression of these control genes is significantly altered by the experimental conditions, this would lead to misinterpretation of the results. However, based on previous studies, beta -actin gene expression has been shown to be stable under many conditions in a variety of different tissues, suggesting that it is a reasonable standard control for most molecular techniques, including RT-PCR and Northern blotting.

For each gene studied by differential hybridization, the question is whether the level of expression is significantly different in the control and experimental state. One approach commonly used to declare differential gene expression is to determine whether its average expression level varies by more than an arbitrarily set value, typically a twofold difference between the control and treatment conditions, but this method lacks convincing biological relevance. Another approach has been to apply the Student's t-test to the expression levels of each gene. This has the potential for addressing some of the shortcomings of the fixed fold-threshold approach; however, in the majority of large microarray studies, the lack of replication of results has precluded the use of such analysis. In the present study, we have chosen to use relatively small and more affordable microarrays, which enabled us to study enough replicates, so that each gene can be compared by t-test.

Examination of the expression of 1,751 genes identified 58 that were differentially expressed in response to small chronic elevations of plasma ANG II. The stringent analytical approach resulted in only ~37% of genes passing the criteria set in the initial screen of the outer medullary tissues, and only 33% of the genes from the inner medullary tissues. Although many other genes may have been differentially expressed, the strategy of the present study was to narrow our focus on genes that reliably passed our rigid criteria of acceptance. This is the first expression array analysis in the renal medulla in response to ANG II stimulation so it is not possible to compare our results with others in the literature. We therefore carried out Northern blot analysis to confirm our results to the extent possible, and we have validated the sequences of most of the differentially expressed genes. The genes randomly selected for the comparisons between these two methods demonstrated a high level of agreement between the two methods (Table 3).

The results indicate that ANG II has profound effects on the function of renal medulla and may play an important physiological role in the regulation of the structure and function in this region of the kidney. The results also demonstrated that ANG II could have distinctively different effects in renal outer medulla and inner medulla. Finally, this study demonstrates the feasibility of using appropriate numbers of replicates in microarray experiments and an experimental design to better define physiological and pathophysiological responses to stimuli.


    ACKNOWLEDGEMENTS

We thank G. R. Slocum for help with scanning microarray slides, R. Berdan and SPS Productions for assistance with computer program development for compiling gene identification files, and R. Cole for assistance with the sequencing. We also thank Drs. A.-P. Zou and A. S. Greene for critical discussion of the experimental protocol and statistical analysis and M. M. Skelton for review of the manuscript.


    FOOTNOTES

This study was supported by National Heart, Lung, and Blood Institute Grants HL-66579, HL-54998, HL-29587, and HL-49219. B. Yuan was supported by American Heart Association Postdoctoral Fellowship 20517Z.

Address for reprint requests and other correspondence: A. W. Cowley, Jr., Dept. of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (E-mail: cowley{at}mcw.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published January 23, 2003;10.1152/ajpregu.00257.2002

Received 10 May 2002; accepted in final form 8 January 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Regul Integr Comp Physiol 284(5):R1219-R1230
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