the renin - angiotensin - aldosterone system ( raas ) plays an important role in the control of cardiovascular and renal homeostasis by regulating vascular tone , 
 blood pressure ( bp ) , and fluid volume [ 1 , 2 ] .
angiotensin ii ( ang ii ) is a 
 physiologically active component of the raas , produced via an enzymatic cascade 
 that begins with angiotensinogen ( agt ) cleaving renin ( ren ) to form angiotensin 
 i ( ang i ) , which is then cleaved by the angiotensin converting enzyme ( ace ) to 
 form ang ii .
ang ii causes vasoconstriction 
 directly by activating ang ii type 1 ( at1 ) receptors on vascular smooth 
 muscle , affects fluid volume via at1 receptor activation in the 
 proximal tubule , resulting in renal sodium and water reabsorption , and plays an 
 important role in the regulation of fluid balance by stimulating aldosterone 
 secretion from the zona glomeruloza of the adrenal glands .
ace inhibitors , ang ii receptor antagonists , and aldosterone receptor antagonists have been used as therapeutic interventions to treat hypertension .
the genes of the renin - angiotensin have been linked to and/or associated with hypertension in animal models and humans .
recently , transgenic rodent models have been developed that over express both human ren and angiotensinogen , which leads to hypertension via chronic overproduction of ang ii .
specific examples include the murine double transgenic line ( ang 204/1 ren 9 ) , which produces a mean arterial bp 40 mmhg higher than background mice ( c57bl/6j ) that lack the human genes .
in addition , transgenic rats harboring the mouse renin-2 gene developed hypertension , cardiac hypertrophy , and renal damage 
 .
the tsukuba hypertensive mice ( thm ) , which express the human ren and angiotensinogen genes , have been proven 
 to develop hypertension .
originally , the raas was viewed solely as an 
 endocrine system , in which angiotensinogen of hepatic origin is 
 secreted intothe systemic circulation and cleaved by ren and ace to 
 produce the active peptide ang ii .
however , there is increasing 
 evidence that suggests a raas may reside within several organs or 
 tissues , including kidney , lung , heart , and vascular smoothmuscle 
 cells ( smc ) , where it is believed to act in a functionally independent paracrine / autocrine fashion .
this 
 hypothesis is further supported by the fact that all components of the raas in the heart , kidney , and lung contain the ace component [ 3 , 6 ] .
additionally , high concentrations of ang ii 
 have been demonstrated in the plasma , heart , and kidney of thm [ 7 , 8 ] . in the kidney , prostaglandins ( pgs )
are important mediators of hemodynamic regulation , salt and water homeostasis , and ren release [ 9 , 10 ] .
the main pg in the kidney is pge2 , which is synthesized from arachidonic acid ( aa ) by enzymatic reactions , 
 particularly cyclooxygenases and prostaglandin e synthases ( pges ) .
cyclooxygenase ( cox ) derived pgs have two distinct membrane - anchored isoenzymes , cox-1 and cox-2 .
cox-1 is constitutively expressed and found in most normal body tissues , while cox-2 is expressed in normal tissues at low levels and is highly induced by proinflammatory mediators in inflammation , injury , and pain settings .
the membrane - associated pges-1 ( mpges-1 ) is inducible and functionally linked to cox-2 , while mpges-2 is constitutive and coupled to both cox isoforms .
it has been suggested that regulation of cox-2 in the kidney is altered by the raas system [ 9 , 12 ] . in thm mice , increased expression of cox-2 in the macula densa has been reported , and an important role for raas in cardiac hypertrophy has been noted .
overexpression of cox-2 has also been observed in the 
 aldosterone - treated animals in normotensive and hypertensive rats . in the lung ,
significant reduction in bp was seen in pg ep1 
 receptor - deficient mice and was accompanied by increased ren - ang activity .
there are currently no published reports on the cellular expression and microanatomic location of cox-1 , cox-2 , mpges-1 , mpges-2 in hypertensive transgenic mice .
therefore , using immunohistochemistry , we investigated the microanatomic location and cellular 
 expression of cox-1 , cox-2 , mpges-1 , mpges-2 in kidney , lung , and heart tissues 
 obtained from renin - angiotensingen transgenic mice .
this study is the first to 
 report on the pulmonary and cardiorenal microanatomic expression of these 
 molecules in this animal model for human hypertension .
15- to 20-week - old male ( n = 15 ) and female ( n = 15 ) double 
 transgenic mice ( h - ang 204/1 h - ren 9 ) were used in the 
 study .
the mice were derived from a founder colony of 5 
 male mice expressing human angiotensinogen ( h - ang 204/1 ) 
 and 6 females expressing human renin ( h - ren6 ) , obtained 
 from dr .
curt sigmund at the university of iowa , school of 
 medicine . at charles river laboratories ( wilmington , massachusetts ) , 
 female mice that expressed human ren
were 
 bred with angiotensinogen - expressing males to produce the 
 double transgenic line .
all the procedures were in compliance 
 with the pfizer ann arbor laboratories animal care 
 and use committee .
lung , kidney , and heart tissues were obtained from the mice 
 at the time of necropsy .
tissues were fixed in 10% neutral 
 buffered formalin for 24 hours and embedded in paraffin 
 wax .
3m - thick sections were then cut and stained immunohistochemically 
 with antibodies to cox-1 , cox-2 , mpges-1 , and mpges-2 . to analyze the expression of cox-1 , cox-2 , mpges-1 , and mpges-2
, 
 3m sections were cut from formalin - fixed , paraffin - embedded blocks , mounted on positively charged glass slides , dried , and then loaded on the automated 
 immunostainer ( room temperature using a ventana discovery ( ventana medical 
 systems , tucson , az ) .
sections were incubated for 30 minutes with serum free dakocytomation protein blocker ( dako corporation , dako , ca ) and then rinsed and incubated for 4 minutes with avidin - biotin blocking solution ( ventana medical systems ) .
antigen retrieval was completed with a ventana specialty solution ( 8 = ph ) ( ventana medical systems ) .
automation included exposure to 100l of primary anti - cox-1 ( 1:200 ) , cox-2 ( 1:20 ) , mpges-1 ( 1:750 ) , or mpges-2
( 1:1000 ) antibody ( cayman chemical , ann arbor , mi ) diluted with reagent diluent ( ventana medical systems ) at room 
 temperature for 60 minutes .
100l of the appropriate anti - rabbit biotinylated 
 igg linking solution ( vector laboratories ) was applied to each section at 1:200 
 dilution for 60 minutes at room temperature .
sections were again rinsed and 
 allowed to react with 100l of diaminobenzidine ( dab detection kit ) 
 substrate solution ( ventana medical systems ) for 8 minutes , followed by 
 counterstaining with hematoxylin and then bluing reagent for 4 minutes each , 
 removed from the autostainer , washed in warm water , dehydrated through graded 
 alcohol , cleared in xylene , and cover slipped . control reactions included ( 1 ) 
 sections incubated with the omission of primary antibody and processed as 
 mentioned above , and ( 2 ) sections incubated with normal rabbit serum instead of 
 the primary antibody and processed as above .
the pulmonary and cardiorenal cellular expression and distribution of cox-1 , cox-2 , 
 mpges-1 , and mpges-2 are summarized in tables 1 , 2 , 3 , and 4 . staining intensity ranged from negative 
 (  ) to strong ( + + + ) . in the kidney ,
strong diffuse cytoplasmic cox-1 expression occurred in the distal convoluted tubules ( dct ) , cortical collecting ducts , and medullary collecting ducts , while proximal convoluted tubules ( pct ) lacked cox-1 expression ( see figure 1(a ) and table 1 ) .
moderate ( + + ) diffuse cox-1 cytoplasmic staining was present in vascular endothelial cells ( ec ) and smc , cortical interstitial cells ( ic ) , the glomerular visceral epithelium , and the capsular parietal epithelium .
mild ( + ) cox-1 expression was present in medullary ic , glomerular podocytes , and the medullary ascending limb ( mal ) .
expression of cox-1 was equivocal in the macula densa . in the lungs and heart ,
cox-1 was strongly expressed in alveolar macrophages , the bronchial and bronchiolar epithelium , and cardiac vascular endothelial cells , but moderately expressed in alveolar septa , bronchial smooth muscle cells , pulmonary vascular endothelial cells , pulmonary vascular ec , and cardiac vascular smc .
cox-1 was not expressed in cardiac myocytes ( see figure 1(b ) and table 2 ) .
mpges-2 was expressed in the kidney at a moderate diffuse level in pct and dct , capsular parietal epithelium , and medullary collecting ducts ( see figure 2(a ) and table 1 ) .
mild expression was present in macula densa , vascular smc and ec , 
 glomerular podocytes and the visceral epithelium , cortical collecting ducts , 
 and the mal .
pulmonary expression of mpges-2 included 
 strong diffuse staining in bronchial and bronchiolar epithelial cells ( see 
 figure 2(b ) and 
 table 2 ) , moderate staining in alveolar macrophages and septa , mild staining in bronchial smc , and pulmonary vascular ec and smc .
renal cox-2 expression was strong in the pct and dct ( see figure 3(a ) and table 3 ) , moderate in macula densa , vascular smc and ec , and medullary collecting ducts , and mild in 
 cortical and medullary interstitial cells , cortical collecting ducts , and the 
 mal .
no expression was present in glomeruli or the capsular ( parietal ) 
 epithelium . in the lung , marked ( + + + ) cox-2 expression was present in alveolar 
 macrophages , and the bronchial and bronchiolar epithelium ( see figure 3(b ) and table 4 ) .
cardiac vascular ec had mild cox-2 expression , while cardiac myocytes and cardiac vascular smc were negative .
for mpges-1 , expression was mild in renal vascular smc and ec , 
 cortical and medullary collecting ducts , and the mal ( see figure 4(a ) and table 3 ) .
expression in the macula densa , glomerular ( visceral ) epithelium , and 
 capsular ( parietal ) epithelium was equivocal .
mpges-1 was the highest in bronchial and 
 bronchiolar epithelial cells ( see figure 4(b ) and table 4 ) .
mild expression was present in alveolar 
 macrophages and alveolar septa , while expression in pulmonary vascular ec and 
 smc was equivocal .
mild mpges-1 expression was present in cardiac vascular ec 
 and cardiac vascular smc , while other cardiac microanatomic locations lacked 
 expression .
pgs are modulators of physiological functions and contribute to ren release , regulation of renal microvascular hemodynamics , salt balance , and bp controlvia mechanisms involving the regulation of vascular tone and 
 renal excretory function .
the kidney is capable of synthesizing all 
 types of pgs , especially pge2 and pgi2 , which influence urinary sodium excretion directly through inhibition of the tubulartransport 
 function and indirectly through the regulation of renin - angiotensin system 
 activity .
cox , a rate - limiting enzyme in the pg biosynthesispathway , exists in two major isoforms : the constitutive cox-1 and the 
 inducible cox-2 .
cox-1 is expressed constitutively at varying levels in the majority of tissues and generally plays a role in tissue homeostasis . in this study ,
the highest degree of renal cox-1 expression 
 occurred in the dct , cortical collecting ducts , and medullary collecting ducts , 
 while mild - to - moderate cox-1 expression occurred in other microanatomic renal 
 locations .
these results are concurrent with cox-1 being the most abundant cox isoform that is constitutively expressed in the kidney and regionally localized in the renal vasculature , collecting ducts , and papillary ic across various species [ 20 , 21 ] .
further evidence supporting these results includes previous work in 
 which various species ( cow , dog , guinea pig , human , monkey , 
 mouse , rabbit , rat , and sheep ) exhibited high levels of cox-1 expression 
 in the collecting ducts [ 20 , 22 , 23 ] . since cox-1 is expressed in the collecting 
 ducts of the nephron , an active area in the regulation of sodium excretion in 
 both laboratory animals and humans , it is not surprising that bp increases in cox-1 deficient mice compared 
 with wild - type controls .
nonselective 
 nonsteroidal anti - inflammatory drugs ( nsaids ) may aggravate renin - independent , 
 sodium - sensitive hypertension , possibly in part by inhibition of the cox-1 
 responsible for sodium excretion . in rats infused with selective cox inhibitors , ns-398 or meloxicam ,
direct renal interstitial volume expansion significantly increased renal interstitial hydrostatic pressure and fractional excretion of sodium .
our finding that most cardiac microanatomic 
 locations had cox-1 expression with the exception of cardiac myocytes is 
 consistent with other studies showing cox-1 expression in both ec and 
 smc of the fetal ductus arteriosus of pigs , rats , sheep , and humans [ 2831 ] . 

furthermore , cox-1 was constitutively expressed in the ec of the aorta and microvasculature , fibrous connective tissue of the tricuspid valve , and chordae tendinae of the heart of dog .
other examples of cox-1 expression in lungs include human lungs , and the bronchiolar epithelium and smooth muscle , alveolar macrophages , ec , and vascular smc of rat . as a result
mpges-2 is constitutively expressed 
 in several tissues , is not induced during inflammation , and has been proposed 
 to mediate pge2 production from both cox isoforms [ 11 , 35 ] . in our study , moderate mpges-2 expression was present in pct and dct , the capsular parietal epithelium , and medullary collecting ducts , while mild or no expression was present in other renal microanatomic locations .
in other studies , high expression of mpges was reported in distal tubules , and medullary collecting ducts in normal mouse kidney .
other 
 studies using primary cultures of neonatal ventricular myocytes have shown that 
 mpges-2 is constitutively synthesized in myocytes and is not regulated .
cox-2 is expressed in variable locations in the kidney across various species . cox-2 deficient mice exhibited severe disruption of renal development and function , suggesting an important role for cox-2 in renal development [ 38 , 39 ] .
the normal rodent and canine kidney have prominent constitutive cox-2 expression in the macula densa and thick ascending limb of loop of henle , whereas cox-2 is absent at these sites in the normal primate and human kidney . in our study , cox-2 was present in all microanatomic renal locations , with the exception of the glomeruli and capsular ( parietal ) epithelium .
this finding is consistent with the known regulatory association between cox-2 and ren reported by schnermann .
demonstration 
 that indomethacin and sc58236 ( a selective cox-2 inhibitor ) can decrease bp and suppress 
 plasma ren activity in rats lends further support to this association [ 41 , 42 ] . 

the marked increase of cox-2 expression in the pulmonary epithelium in 
 our study is consistent with the known role of respiratory epithelia as a first 
 line of defense .
pulmonary 
 epithelial cells have an important role in airway homeostasis , perform many 
 important biological functions , and represent the first line of defense against 
 infection .
activation of the renin - angiotensin system has also been 
 demonstrated in acute asthma .
cardiac vascular ec and smc had cox-2 
 expression , while no expression was present in cardiac myocytes or smc in our 
 study .
an important role for ang ii in the regulation of cox-2 has 
 been reported .
mpges-1 
 was present in renal vascular ec and smc , cortical and medullary collecting 
 ducts , mal , all pulmonary microanatomic locations , and cardiac vascular ec and 
 smc , while no expression was present in other renal or cardiac microanatomic 
 locations .
these findings are consistent with increased mpges expression 
 described in patients with hyperprostaglandin e syndrome .
mpges-1 
 was also induced by the proinflammatory cytokine in cardiac myocytes and 
 fibroblasts . in 
 conclusion , in hypertensive mice there are ( a ) significant microanatomic variations in the pulmonary , renal , and 
 cardiac distribution and cellular localization of mpges-1 , mpges-2 , 
 cox-1 , and cox-2 and ( b ) no 
 differences in expression between genders .
 hypertensive mice that express the human renin and angiotensinogen genes are used as a model for human hypertension because they develop hypertension secondary to increased renin - angiotensin system activity . 
 our study investigated the cellular localization and distribution of cox-1 , cox-2 , mpges-1 , and mpges-2 in organ tissues from a mouse model of human hypertension . 
 male ( n = 15 ) and female ( n = 15 ) double transgenic mice ( h - ang 204/1 h - ren 9 ) were used in the study . 
 lung , kidney , and heart tissues were obtained from mice at necropsy and fixed in 10% neutral buffered formalin followed by embedding in paraffin wax . 
 cut sections were stained immunohistochemically with antibodies to cox-1 , cox-2 , mpges-1 , and mpges-2 and analyzed by light microscopy . 
 renal expression of cox-1 was the highest in the distal convoluted tubules , cortical collecting ducts , and medullary collecting ducts ; while proximal convoluted tubules lacked cox-1 expression . 
 bronchial and bronchiolar epithelial cells , alveolar macrophages , and cardiac vascular endothelial cells also had strong cox-1 expression , with other renal , pulmonary , or cardiac microanatomic locations having mild - to - moderate expression . 
 mpges-2 expression was strong in the bronchial and bronchiolar epithelial cells , mild to moderate in various renal microanatomic locations , and absent in cardiac tissues . 
 cox-2 expression was strong in the proximal and distal convoluted tubules , alveolar macrophages , and bronchial and bronchiolar epithelial cells . 
 marked mpges-1 was present only in bronchial and bronchiolar epithelial cells ; while mild - to - moderate expression was present in other pulmonary , renal , or cardiac microanatomic locations . 
 expression of these molecules was similar between males and females . 
 our work suggests that in hypertensive mice , there are ( a ) significant microanatomic variations in the pulmonary , renal , and cardiac distribution and cellular localization of cox-1 , cox-2 , mpges-1 , and mpges-2 , and ( b ) no differences in expression between genders . 
1. INTRODUCTION
2. MATERIALS AND METHODS
3. RESULTS
4. DISCUSSION