Neonatal Losartan Treatment Suppresses Renal Expression of Molecules Involved in Cell-Cell and Cell-Matrix Interactions
Yun Chen*,
Daina Lasaitiene*,
Britt G. Gabrielsson,
Lena M.S. Carlsson,
Håkan Billig*,
Björn Carlsson,
Niels Marcussen,
Xiao-Feng Sun and
Peter Friberg*
*Department of Physiology, University of Gothenburg, and RCEM, Department of Internal Medicine, Sahlgrenska University Hospital, Gothenburg, Sweden; Institute of Pathology, Aarhus Kommunehospital, Aarhus, Denmark; and Department of Oncology, Institute of Biomedicine and Surgery, University of Linköping, Linköping, Sweden
Correspondence to Dr. Yun Chen, Department of Physiology, University of Gothenburg, Box 432, SE 405 30 Gothenburg, Sweden. Phone: 46-31-7733557; Fax: 46-31-7733512; E-mail: yun.chen{at}kidney.med.gu.se
ABSTRACT. Lack of neonatal angiotensin II type 1 receptor (AT1)stimulation produces renal abnormalities characterized by papillaryatrophy and impaired urinary concentrating ability, but themechanisms involved are still unclear. DNA microarray was usedto identify genes that are differentially expressed in renalmedulla in response to neonatal treatment with AT1 receptorantagonist losartan (30 mg/kg per d), which commenced within24 h after birth. The data showed that losartan treatment for48 h downregulated 68 genes, 30% of which encode various componentsof cytoskeleton and cytoskeleton-associated proteins, extracellularmatrix, and enzymes involved in extracellular matrix maturationor turnover. With the use of immunohistochemistry and Westernimmunoblot, the microarray data were confirmed and it was demonstratedthat losartan suppressed renal expression of syndecan 2, -smoothmuscle actin, MHC class II, and leukocyte type 12-lipoxygenaseby day 4. In addition, losartan inhibited medullary expressionof integrin 6 and caused relocalization of integrins 6 and 3.Moreover, losartan inhibited cell proliferation in medullarytubules by day 9, as detected by Ki-67 immunostaining. Thisstudy provides new data supporting the contention that a lackof AT1 receptor stimulation results in abnormal matrix assembly,disturbed cell-cell and cell-matrix interactions, and subsequentabnormal tubular maturation. Moreover, regulation of the expressionof leukocyte type 12-lipoxygenase and -smooth muscle actin bythe renin-angiotensin system in the immature kidney adds newknowledge toward the understanding of renal vascular development.
Renal maldevelopment in premature infants and infants who aresmall for gestational age is known to be related to hypertensionin adult life (1) and may be attributed to the suppressed intrarenalrenin-angiotensin system (RAS) in neonates (2). In humans, nephrogenesisis completed before gestational week 36, whereas, in rodents,nephrogenesis is not completed until 10 d after birth (3). Thus,rats and mice are born with immature kidneys, and the first2 postnatal weeks correspond approximately to the second andthird trimesters in humans. This difference provides a convenientanimal model for studying mechanisms underlying the RAS-mediatedkidney development in human fetus.
The importance of the RAS for normal kidney development hasbeen demonstrated by a number of studies using gene targetingor pharmacologic interruption of the RAS (4). Thus, inhibitionof angiotensin-converting enzyme activity or angiotensin IItype 1 receptor (AT1) but not AT2 signaling in animals withan ongoing nephrogenesis induces kidney abnormalities (5). Themost pronounced structural change is papillary atrophy, andit is associated with impaired urinary concentrating ability(5). Kidney vasculature is also affected, characterized by fewer,thicker, and shorter afferent arterioles (6).
Despite the well-recognized renal abnormalities after neonatalRAS inhibition, little is known about mechanisms by which alack of AT1 stimulation leads to irreversible renal damage.Thus, the first aim of the present study was to use DNA microarrayto identify genes involved in the RAS-mediated developmentalprocess of renal medulla, a region where the predominant structural-functionaldefects are found in rats subjected to neonatal RAS inhibition.In developing renal medulla, the medullary rays and blood vesselshave been shown to express AT1 receptors (7,8). We used theAT1 receptor antagonist losartan because we wanted to studyexpression of genes related specifically to the AT1 receptor.Our results showed that neonatal losartan treatment downregulated68 genes and upregulated only nine genes in renal medulla.
The second aim was to validate the microarray data using immunohistochemistryand Western immunoblot. Because 30% of the downregulated genesencode molecules involved in cell-matrix interactions, we furtheranalyzed key molecules syndecan 2, integrin 3, and 6, whichare important for cell-matrix interactions (9–11). Furthermore,three of the downregulated genes—-smooth muscle actin(-SMA), MHC class II (MHC II), and leukocyte type 12-lipoxygenase(12-LO)—were examined further at the protein level. MHCII has been shown to be expressed by renal tubular epithelialcells during tubulogenesis (12). -SMA has been shown to be expressedby developing kidney vasculature (13), whereas 12-LO metabolizesarachidonic acid to produce bioactive lipid 12(S)-hydroxyeicosatetraenoicacid, an important mediator of the effects of angiotensin IIon vascular structure and function in adults (14–17).Given the tubular and vascular changes induced by neonatal RASblockade (18,19), the identified molecules may provide new insightinto the mechanisms involved in the RAS-mediated kidney development.Finally, we wanted to investigate whether neonatal AT1 receptorblockade would affect renal growth, by determining proliferationand apoptosis.
Animals
Within 24 h after birth, Wistar rats (n = 5 per group) weretreated intraperitoneally with losartan (2 x 15 mg/kg per d)or isotonic saline (2 x 10 ml/kg per d). Two days later, animalswere anesthetized with pentobarbital (60 mg/kg) and kidneyswere extirpated. This time point was chosen on the basis ofour recent finding demonstrating renal structural abnormalities,evidenced as tubular dilation, already at such an early timeafter starting treatment with enalapril (20). Renal medullawas dissected from the cortex, snap-frozen in liquid nitrogen,and used for subsequent RNA preparation.
In a second series of experiments, rats (n = 8 per group) weretreated with saline or losartan and kidneys were collected after2, 4, or 9 d of treatment. One kidney was dissected into cortexand medulla, snap-frozen in liquid nitrogen, and used for Westernimmunoblot. The other kidney was divided into two pieces bycrosscut, and one half was fixed in 4% formaldehyde and theother half was embedded in Tissue-Tek OCT compound and snap-frozenin liquid nitrogen. Paraffin sections (3 µm) or cryosections(5 µm) were prepared for immunohistochemistry. For morphologicevaluation, paraffin sections were stained with hematoxylinand eosin. The rats were kept at the local animal departmentwith free access to normal rat diet and tap water, controlledroom temperature of 24°C, and 12-h dark/light cycle. Theexperiments were approved by the animal ethics committee inGothenburg, Sweden.
RNA Preparation
Total RNA was prepared (pooled renal medulla from five pups)with a Qiagen RNeasy mini-kit according to the manufacturer’sinstructions. The concentration of total RNA was determinedby the optical density at 260 nm, and its purity was judgedby the 260/280 ratio. The integrity of the RNA was examinedon a 1% agarose gel containing 2.2 M formaldehyde.
Preparation of cRNA and Gene Chip Hybridization
Double-strand cDNA was generated from 8 µg of RNA usingreverse transcription primer containing poly (dT) and a T7 RNApolymerase promotor sequence (Superscript Choice System, LifeTechnologies). Synthesis of biotin-labeled cRNA was carriedout by in vitro transcription using ENZO BioArray HighYieldRNA transcription labeling kit (Affymetrix). Triplicate cRNAwere prepared from the RNA sample of saline-treated rats andduplicate cRNA from that of losartan-treated rats. These fivecRNA preparations were purified, fragmented, and hybridizedto five Affymetrix GeneChip arrays (RG_U34A) containing 8784probe sets (77% known genes and 23% expressed sequence tags).The arrays were washed and stained with streptavidin-phycoerythrinconjugate and scanned by the Hewlett-Packard GeneArray Scanner.For enhancing the signals, the arrays were stained further withanti-streptavidin antibody for 30 min followed by a 15-min stainingwith a streptavidin-phycoerythrin conjugate.
Data Analysis
The scanned output files were inspected visually for hybridizationartifacts before being analyzed by the GeneChip 3.1 software(Affymetrix). For computing comparisons of gene expression betweentwo microarrays, the DNA microarrays were globally scaled toan average intensity of 500. Comparisons were made between thetriplicate DNA microarrays used for analysis of the saline-treatedgroups and the duplicate DNA microarrays used for analysis ofthe losartan-treated group, generating a total of six comparisonfiles. Genes whose expression differed in the losartan-treatedcompared with the saline-treated animals were identified bythe "difference call" (Diff Call) algorithm developed by Affymetrix(GeneChip 3.1). With the Diff Call, the gene expression is classifiedas increased (I), marginally increased (MI), no change (NC),decreased (D), or marginally decreased (MD) using defaultedvalues (Affymetrix) for the thresholds that define the boundariesamong I, MI, NC, MD, and D. A Diff Call of D or MD was scored–2 or –1, respectively; a Diff Call of I or MI wasscored 2 or 1, respectively. The general criteria used to selectgenes with increased expression in the losartan-treated comparedwith the saline-treated group from these six comparisons wereas follows: a sum of Diff Call scores >8 (minimum four ofthe six comparisons classified as I), an average fold change>1.5, and a signal (Average Difference; Affymetrix) >50.Conversely, criteria for selection of genes with decreased expressionwere a sum of Diff Call scores <–8, an average foldchange <–1.5, and a signal >50.
Flow Cytometry for S-Phase Fraction Analysis
Rats (n = 8 per group) were treated with saline or losartanfor 4 d before they were killed and their kidneys were collected.The kidneys were treated with 3 mg/100 ml trypsin (Sigma, St.Louis, MO) in a citrate buffer for 10 min at room temperature.After filtration through a nylon mesh, the suspension was stainedwith propidium iodide. The cell suspensions were analyzed witha FACScan flow cytometer (Becton-Dickinson). For each sample,15,000 cells were counted. S-phase fraction (SPF) was estimatedby using a rectangular model. The number of S-phase cells wascalculated by multiplying the number of channels between theG0/1 and G2/M peaks by the mean number of cells per channelin a part of the S-phase interval judged as representative bythe operator. Small disturbing peaks in the S-phase region couldbe excluded when the SPF was calculated.
Immunohistochemistry
Immunostaining for syndecan 2, integrin 3, -SMA, and MHC IIwas performed on cryosections, whereas immunostaining for integrin6 was performed on paraffin sections. Sections were incubatedwith primary antibodies (Table 1), followed by a correspondingsecondary antibody (1:100). Binding of peroxidase-labeled secondaryantibody was detected by incubation with substrate 3-amino-9-ethyl-carbazole(Sigma), containing H2O2. Sections were counterstained withMayer’s hematoxylin.
Table 1. List of primary antibodies used in this studya
For detection of proliferation and apoptosis, paraffin sectionswere used. Proliferating cells were identified by incubatingsections with anti-Ki-67 antibody, followed by a secondary antibody(1:100). Apoptosis was determined by in situ ligation of thebiotinylated oligonucleotide hairpin probe (HPP) with one nucleotideoverhang in the 3' end, followed by incubation with avidin-biotin-peroxidasecomplex (Vector Laboratories, Burlingame, CA), according tothe procedure described previously (21). Visualization was performedusing diaminobenzidine solution (0.25% diaminobenzidine, 0.01%H2O2, and 0.04% NiCl) with methyl green counterstaining. Forquantifying the number of proliferating or apoptotic cells,renal cortex and medulla was delineated according to Kriz andBankir (22). Vision fields with a frame of 3070 µm2 weresystematically sampled with the use of a motorized stage. Thenumber of Ki-67-positive or HPP-positive nuclei in the tubulesor interstitium and of total nuclei within the frame was countedat 40x magnification. The data were expressed as a percentageof total nuclei. The number of total nuclei counted was 562± 82 per section. Negative controls were obtained byreplacing the primary antibodies with 1% BSA or omitting theT4 DNA ligase (for HPP).
Western Immunoblot
Tissues were homogenized in buffer that contained 250 mM sucrose,10 mM HEPES-Tris (pH 6.95), and protease inhibitors (CompleteMini, Roche). Protein concentration was measured using a Bio-RadProtein Assay kit. Aliquots of protein were solubilized in Laemmlisample buffer and separated by electrophoresis on 4–15%Tris-HCl gradient gel (Bio-Rad, Hercules, CA). Proteins weretransferred to Hybond-P membrane (Amersham). After blockingin 5% nonfat milk with TBS-T [20 mM Tris (pH 7.6), 137 mM NaCl,0.1% Tween 20], the membrane was incubated with a primary antibody(Table 1). The proteins were detected using peroxidase-linkedsecondary antibody and enhanced chemiluminescence detectionsystem (Amersham) according to enhanced chemiluminescence Westernblotting protocols. Nonspecific binding was evaluated by replacingthe primary antibody with normal serum or IgG. Bands were visualizedusing a Fuji LAS-1000 cooled CCD camera/Dark Box, using theImage Reader LAS-1000 v1.1 software, and the density of thebands was analyzed using the Image Gauge software v3.45. Asboth microarray data and Western immunoblot data did not revealany difference in the expression of -actin mRNA and proteinbetween saline-treated and losartan-treated groups (data notshown), -actin was used as internal control and protein levelwas expressed as a ratio to -actin.
Statistical Analyses
All values are expressed as means ± SD. Statistical comparisonswere made according to two-tailed t test for unpaired data.P < 0.05 was considered significant.
Morphology
Medullary tubular dilation was apparent at 2 d after initiationof losartan treatment (Figure 1B). By day 4, cortical tubulardilation was found (Figure 1D). Papillary atrophy in losartan-treatedrats was seen at day 9 (Figure 1F).
Figure 1. Sequential development of the renal papilla in the control (A, C, and E) and losartan-treated rats (B, D, and F). Note that medullary tubular dilatation (arrow) is apparent at 2 d after initiation of losartan treatment (B). By day 4, cortical tubular dilation (arrows) is observed (D). Papillary atrophy in losartan-treated rats is seen already at day 9 (F).
DNA Microarray Data
Neonatal losartan treatment downregulated 68 genes, 30% of whichencode various components of cytoskeleton and cytoskeleton-associatedproteins, extracellular matrix (ECM), and enzymes involved inECM maturation or turnover Table 2).
Table 2. Downregulated genes encoding components of cytoskeleton and cytoskeleton-associated proteins, ECM, enzymes involved in ECM maturation or turnover, and MHC family and leukocyte type 12-lipoxygenasea
Renal Expression of Syndecan 2, Integrin 3, and Integrin 6
To investigate whether suppression of these genes would resultin disturbed cell-matrix interaction, we determined renal expressionof key molecules, syndecan 2, and integrins 6 and 3, which areimportant for cell-matrix interaction (9–11).
Syndecan 2 was expressed mainly by tubular structures (Figure 2B).Western immunoblot analysis revealed that the protein levelof syndecan 2 was lower in both renal cortex and medulla fromlosartan-treated 4-d-old rats than those from controls (Figure 2A),thus confirming the microarray data (Table 2).
Figure 2. Expression and localization of syndecan 2 in the renal cortex and medulla. (A) A specific 90-kD band was recognized by anti-syndecan 2 antibody. Density analysis of the 90-kD bands shows a significantly decreased level of syndecan 2 in the cortex and medulla from the losartan-treated rats (n = 7 per group). **P < 0.01 and ***P < 0.001 versus saline. (B) Immunostaining of syndecan 2 is found in the tubular epithelial cells.
Losartan treatment for 4 d decreased the protein level of integrin6 in the renal medulla but not in the cortex (Figure 3A). Inthe control rats, basolateral immunostaining of integrin 6 wasobserved in tubules (Figure 3B). Losartan altered this distributionpattern, and apical immunostaining of integrin 6 was observedin the dilated cortical tubules (Figure 3B).
Figure 3. Expression and localization of integrin 6 in the renal cortex and medulla. (A) A specific 100-kD band was recognized by anti-integrin 6 antibody. Density analysis of the 100-kD bands shows a significantly decreased level of integrin 6 in the medulla but not cortex from the losartan-treated rats (n = 5–8 per group). *P < 0.05 versus saline. (B) Basolateral immunostaining of integrin 6 is observed in the renal tubules from the control rats. Losartan treatment altered this distribution pattern, and the dilated cortical tubules show apical staining of integrin 6.
Immunostaining of integrin 3 was found in the glomeruli anddistal nephrons (Figure 4). In the tubular epithelial cellsof the control rats, the staining was focused to the basal membrane(Figure 4, A and B). Losartan treatment for 4 d resulted indiffused integrin 3 staining (Figure 4, C and D).
Figure 4. Expression and localization of integrin 3 in the renal cortex and medulla. Immunostaining of integrin 3 is found in the glomeruli and distal nephrons. The staining is localized to the basal membrane of tubular epithelial cells and is compact in the control rats (A and B). Losartan treatment for 4 d results in diffused integrin 3-staining (C and D).
Renal Expression of MHC II, -SMA, and 12-LO
To validate further the microarray data, we determined the proteinlevel of three of the downregulated genes revealed by microarray,namely MHC II, -SMA, and 12-LO. Immunohistochemistry demonstratedMHC II staining in tubules of the medulla and cortex and fewinterstitial cells in the cortex in normal kidneys on day 4(Figure 5). Remarkably, the tubular staining of MHC II was absentin the renal medulla and cortex from losartan-treated rats (Figure 5).
Figure 5. Expression and localization of MHC class II (MHC II) protein in the renal cortex (A and C) and medulla (B and D). Kidney sections from 4-d-old rats treated with saline vehicle (A and B) or losartan (C and D) from birth until the day that the rats were killed. The tubular staining is evident in normally developing kidneys (A and B), whereas it is absent in the kidneys of rats subjected to losartan (C and D).
Western immunoblot revealed decreased expression of -SMA inthe renal medulla from the losartan-treated rats on day 4 (Figure 6A).Immunostaining of -SMA was localized to the renal arteriolesand peritubular capillaries (Figure 6B). Using Western immunoblot,we found that losartan treatment for 4 d decreased the proteinlevel of 12-LO in the renal medulla (Figure 7).
Figure 6. Expression and localization of -smooth muscle actin (-SMA) in the renal cortex and medulla. (A) A specific 42-kD band was recognized by anti--SMA antibody. Density analysis of the 42-kD bands shows a significantly decreased level of -actin in the medulla from the losartan-treated rats (n = 8 per group). *P < 0.05 versus saline. (B) Immunostaining of -SMA is localized to the renal arterioles and peritubular capillaries.
Figure 7. Western immunoblot analysis of leukocyte type 12-lipoxygenase (12-LO) in the renal medulla and cortex from 4-d-old rats treated with saline vehicle or losartan from birth until the day that the rats were killed. A specific 75-kD band was recognized by anti-12-LO antibody. Density analysis of the 75-kD bands shows a significantly decreased level of 12-LO in losartan-treated rats (n = 8 per group). *P < 0.05 versus saline.
Proliferation and Apoptosis
To investigate whether neonatal AT1 receptor blockade wouldaffect renal growth, we studied cell proliferation by determiningSPF of renal cells with flow cytometry. Neonatal losartan treatmentfor 4 d did not change renal cell SPF (5.39 ± 1.06% versus5.96 ± 1.5% in controls). Because this assay was performedon whole-kidney homogenates, we might have missed local changescaused by neonatal losartan treatment. To evaluate this, weused immunohistochemistry to study the spatiotemporal patternof proliferation and apoptosis, with the help of anti-Ki-67antibody and HPP, respectively. Quantification of the Ki-67-positivecells revealed that cell proliferation was not changed untilday 9, when losartan-treated rats had a significantly lowernumber of proliferating cells in the medullary tubules but notin the interstitium, compared with the controls (Figure 8).In the cortex, the number of the Ki-67-positive cells was notchanged by losartan. Moreover, neonatal losartan treatment didnot change the number of apoptotic cells (data not shown).
Figure 8. Effect of neonatal AT1 receptor blockade on renal cell proliferation. (A) Quantification of the Ki-67-positive nuclei in the renal medulla and cortex from 4- and 9-d-old rats. Losartan treatment for 9 d decreases the number of proliferating cells in the medullary tubules (n = 5–8 per group). *P < 0.05 versus saline. (B) Immunostaining of Ki-67 in the renal medulla from 9-d-old rats treated with saline vehicle or losartan from birth until the day that the rats were killed. Immunoreactive Ki-67 is present mainly in the tubular epithelial cells.
The present study demonstrates that neonatal losartan treatmentfor 2 d downregulates genes encoding various components of cytoskeletonand cytoskeleton-associated proteins, ECM, and different enzymesinvolved in ECM maturation or turnover. Moreover, neonatal losartantreatment suppresses renal expression of syndecan 2 and integrin6 and causes relocalization of integrin 6 and integrin 3. Ourdata suggest that the RAS is important for normal establishmentof cell-cell and cell-matrix interactions during kidney development.This is consistent with our recent findings that neonatal RASblockade for 2 d disturbs medullary tubulogenesis and downregulatesE-cadherin expression in dilated medullary tubules (20).
The ECM acts as a physical scaffold for the attachment and organizationof cellular structures (23). The concomitant downregulationof different constituents of the physical scaffold, such astype I and type III collagens, decorin, and lumican, as demonstratedin this study, points to possible changes in collagen fibrilassembly. Indeed, interactions between the fibril-associatedsmall proteoglycans and fibrillar collagen regulate collagenfibrillogenesis, which is one of the major events responsiblefor stabilization of tissue structure (24). Moreover, collagenfibrillogenesis could be affected further by both downregulated"matricellular" protein SPARC, which is highly regulated duringdevelopment and has a collagen-binding capacity (23), and downregulatedlysyl oxidase, which is a key regulator of collagen maturation(25). Taking into account that angiotensin II may regulate theexpression of a variety of genes involved in ECM maintenance(26) and that cultured renomedullary interstitial cells respondto AT1 receptor stimulation with synthesis of ECM components(27), we suggest that interrupted AT1 signaling in developingkidney alters the ECM composition and structure, which are crucialto tissue integrity (28).
The information encoded in ECM is revealed by the interactionbetween individual ECM components and their respective receptorson the cell surface, such as integrins and syndecans (cell surfaceproteoglycans). The present study showed that neonatal blockadeof AT1 receptors decreased renal expression of syndecan 2 andintegrin 6 and caused relocalization of integrins 6 and 3. Interestingly,it has been shown that function-blocking antibody against integrin6 blocks kidney tubulogenesis in vitro (9), whereas kidneysof integrin 3-deficient newborn mice have fewer collecting ductswithin the inner medulla (10). Moreover, relocalization of integrinsfrom the basolateral membrane to the apical membrane has beendescribed in cultured renal tubular epithelial cells subjectedto oxidative stress, which results in weakened epithelial cellattachment to ECM (29). Syndecan 2 is normally present on alladherent cells and participates in ECM assembly, regulatingthe signaling of heparan sulfate binding growth factors (11).It is developmentally regulated and peaks during tissue morphogenesis(30). Hence, it is possible that the downregulation of syndecan2 and integrin 6 and relocalization of integrins 6 and 3 couldresult in impaired cell-ECM interactions and subsequent ECM-mediatedcytoskeletal organization, which are important for several cellfunctions, such as cell proliferation, migration, differentiation,and survival (23). This line of reasoning is supported by ourfinding of concomitant downregulation of various componentsof the cytoskeleton (Table 2).
Collectively, neonatal RAS inhibition caused a gene downregulationof components of cytoskeleton and ECM in developing kidney.This may lead to inadequate matrix assembly and disturbed cell-celland cell-matrix interactions (Figure 9), which are known tobe critical for kidney development. These data provide mechanisticsupport to the notion that an intact RAS is essential for thestructural arrangement of the renal medulla.
Figure 9. Illustration of cell-cell and cell-matrix contacts: actomyosin bundles, cytoskeleton-associated proteins, cell surface proteoglycan, extracellular matrix (ECM), and enzymes for ECM maturation/turnover. Listed molecules are those with decreased mRNA expression caused by neonatal losartan treatment.
In contrast to the very early changes in the tubular structure,i.e., tubular dilation on day 2 (20), the present study demonstratedthat cell proliferation was not affected until day 9, when neonatallosartan treatment inhibited cell proliferation in the renalmedulla. It is possible that the inhibited growth is secondaryto disrupted matrix assembly and/or disturbed cell-cell andcell-matrix interactions (vide supra). It is interesting thatthe inhibited proliferation was confined to the tubular epithelialcells, whereas proliferation in the interstitium was not affected.In accordance with our observation, McCausland et al. (18) reportedthat enalapril treatment from postnatal day 3 to 10 reducescell proliferation in the medullary rays. Thus, neonatal AT1receptor blockade impairs tubular growth and maturation, andthis is in line with the fact that neonatal RAS blockade causesabnormalities in tubular structure and function (5,31). Becausethe changes in proliferation are modest and cannot by themselvesentirely account for the hypoplastic papillary phenotype, onemay speculate that neonatal RAS blockade increases cell death.However, our data showed that neonatal losartan treatment for4 and 9 d had no effect on apoptosis. It is interesting thatwe have recently shown that neonatal enalapril treatment for9 d induces inflammatory changes in the kidney (20), which couldbe due to increased necrosis. Necrosis, however, was not studiedin the present study because of the lack of a proper detectiontechnique.
Keeping in mind the perturbed tubulogenesis in neonatally RAS-inhibitedrats, the observed downregulation of MHC II is of interest becauseMHC II was shown previously to be expressed mainly by tubularepithelial cells of immature kidney (12). The function of MHCII, expressed by developing tubules, is unclear. Besides itsclassical antigen-presenting function, MHC II has been shownto play a role in cell-to-cell recognition and tissue organization(32). Because MHC II expression correlates highly with cellulardifferentiation (33), we propose that interrupted AT1 signalingcould hamper the postnatal differentiation of tubular epithelialcells, as evidenced by abolished expression of MHC II in thesecells, and, therefore, disturb tubulogenesis.
Another finding of the present study is that 12-LO is expressedby immature kidney and the renal expression of 12-LO is regulatedby the RAS. 12-LO metabolizes arachidonic acid to produce bioactivelipid 12(S)-hydroxyeicosatetraenoic acid (HETE). It is interestingthat 12(S)-HETE is an important mediator of the effects of angiotensinII on vascular structure and function in adults (14,34). Moreover,12(S)-HETE seems to be critically involved in angiogenesis (35),a process that is needed for kidney vascularization (7). Itis tempting to speculate that decreased expression of such avasoactive substance during kidney development, as demonstratedby both microarray and Western immunoblot, may disturb renalvascular development. This notion is supported by our observationof the decreased renal expression of -SMA after neonatal RASinhibition because immunohistochemistry revealed -SMA expressionin the renal vasculature of the immature kidney. Indeed, neonatalRAS inhibition causes irreversible abnormalities in the renalarterial vasculature characterized by wall thickening and reducedbranching (6,36).
This study provides new data supporting the contention thata lack of AT1 receptor stimulation results in abnormal matrixassembly, disturbed cell-cell and cell-matrix interactions,and subsequent abnormal tubular maturation as evidenced by decreasedproliferation and loss of MHC II expression by epithelial cells.Regulation of the expression of 12-LO and -SMA by the RAS inimmature kidney, however, adds new knowledge toward our understandingof renal vascular development.
Acknowledgments
This study was supported by the Swedish Medical Research Council(grants 9047, 11285, and 13141), the Medical Society of Gothenburg,and the Swedish Medical Society. Dr. Chen is a recipient ofa postdoctoral fellowship from Wallenberg’s foundation.
Part of this work was presented in abstract form at the 11thEuropean Meeting on Hypertension, June 2001.
The monoclonal anti-integrin 3 antibody developed by Dr. L.Reichardt was obtained from the Developmental Studies HybridomaBank developed under the auspices of the National Instituteof Child Health and Human Development and maintained by TheUniversity of Iowa, Department of Biologic Sciences (Iowa City,IA).
We are grateful to Ms. Margareta Jernås for technicalassistance. We thank Dr. Changlian Zhu for providing HPP andfor discussions.
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Received for publication April 29, 2003.
Accepted for publication January 29, 2004.
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Abstract
Introduction
30% of which encode various components of cytoskeleton and cytoskeleton-associated proteins, extracellular matrix, and enzymes involved in extracellular matrix maturation or turnover. With the use of immunohistochemistry and Western immunoblot, the microarray data were confirmed and it was demonstrated that losartan suppressed renal expression of syndecan 2, 
-smooth muscle actin, MHC class II, and leukocyte type 12-lipoxygenase by day 4. In addition, losartan inhibited medullary expression of integrin 
-actin mRNA and protein between saline-treated and losartan-treated groups (data not shown), 
