Cycloxygenase-2 Is Expressed in Vasculature of Normal and Ischemic Adult Human Kidney and Is Colocalized with Vascular Prostaglandin E2 EP4 Receptors
Karina L. Therland*,
Jane Stubbe*,
Helle C. Thiesson*,
Peter D. Ottosen,
Steen Walter,
Grith L. Sørensen,
Ole Skøtt* and
Boye L. Jensen*
*Department of Physiology and Pharmacology, Department of Immunology and Microbiology, University of Southern Denmark, and Institute of Pathology, Department of Urology, Odense University Hospital, Odense, Denmark.
Correspondence to Dr. Boye L. Jensen, Department of Physiology and Pharmacology, University of Southern Denmark, Winsløwparken 21, 3, DK-5000, Odense C, Denmark. Phone: +45-65-50-37-96; Fax +45-66-13-34-79; E-mail bljensen{at}health.sdu.dk
ABSTRACT. The study was performed to elucidate the distributionand cellular localization of cyclooxygenase (COX)-2 in humankidney and to address localization of downstream targets forCOX-derived prostanoids. Cortex and outer and inner medullatissue were obtained from control kidneys (cancer specimens),kidneys with arterial stenosis, and kidneys of patients whoreceived angiotensin II inhibition or acetylsalicylic acid.Ribonuclease protection assay and Western blot test revealedthat COX-1 and –2 mRNA and protein were expressed in allregions of human kidney (mRNA ratio, cortex:outer medulla:innermedulla COX-1 1:3:20 and COX-2 1:1:3). In adult kidney, immunohistochemicallabeling for COX-2 was associated with smooth muscle cells inpre- and postglomerular vessels and with endothelium, particularlyin vasa recta and medullary capillaries. Western blot test confirmedCOX-2 expression in renal artery. COX-2 had a similar localizationin fetal kidney and was additionally observed in Henle’sloop and macula densa. Human tissue arrays displayed COX-2 labelingof vascular smooth muscle in multiple extrarenal tissues. VascularCOX-2 expression was significantly increased in kidneys witharterial stenosis. COX-1 was colocalized with microsomal prostaglandinE2 synthase (PGES) in collecting ducts, and PGES was also detectedin macula densa cells. Vascular COX-2 was colocalized with prostaglandinE2 EP4 receptors but not with EP2 receptors. Thus, renovascularCOX-2 expression was a constitutive feature encountered in humankidneys at all ages, whereas COX-2 was seen in macula densaonly in fetal kidney. Vascular COX-2 activity in human kidneyand extrarenal tissues may support blood flow and affect vascularwall-blood interaction.
A rate-limiting step in prostaglandin formation is catalyzedby cyclooxygenase (COX). Two isoforms are recognized: constitutiveCOX-1 and inducible COX-2. Selective COX-2 antagonists haverecently been developed and are in widespread clinical use.In contrast to expectations, COX-2 selective blockers exhibitadverse effects related to kidney and cardiovascular function;they have antinatriuretic properties; they lower GFR and RBFand can lead to acute renal failure; and they aggravate preexistinghypertension and may lead to adverse cardiovascular outcomes(1–6). Thus, the data indicate important physiologic rolesfor COX-2 in human cardiovascular and renal homeostasis, butthe renal correlates of these clinical observations remain poorlyunderstood. Both COX genes are expressed in human kidney (7,8),but the quantitative relation of COX-2 to COX-1 expression isnot known. Whether there are differences in expression levelover time or in kidney regions is also not known. Data on thecellular localization of COX-2 in human kidney are inconsistent.
COX-2 has been observed in thick ascending limb of Henle’sloop (cTAL), including the macula densa in fetal human kidney(8–10) and in the kidneys of children with Bartter’ssyndrome (11), a location first described in rat kidney (12).Several studies did not detect COX-2 in the cTAL or macula densaof adult human kidneys (7,8,10), whereas other reports showedan age- or disease-dependent expression of COX-2 in macula densa(11,13,14) and a functional role of COX-2 for stimulation ofrenin secretion in humans (15,16). COX-2 has also been observedin renal vascular tissue (7–9), and observations indicatethat intact prostaglandin synthesis is important for maintenanceof renal function during renovascular hypertension with renalarterial stenosis (17–19). It is not known whether thisdependence involves COX-2 activity.
Three major objectives of the study presented here were to determinethe quantitative regional distribution and cellular localizationof COX isozymes in normal adult human kidney and compare tofetal kidney; to assess COX-2 in human kidneys with arterialstenosis and after pharmacologic interventions; and to elucidatecolocalization of COX enzymes with downstream target molecules.We used quantitative mRNA and protein assays in combinationwith immunohistochemical analysis to address these issues inhuman nephrectomy samples.
Human Tissue Samples
All patients gave written informed consent to participate inthe study. The study was approved by the local ethics committee.None of the cancer patients had received chemotherapy or radiationtherapy before nephrectomy. Samples of normal kidney tissuewere obtained from unaffected areas in nephrectomy specimenswith tumors. Four kidneys with artery stenosis were obtainedduring the 3-yr sampling period. Kidneys were extirpated atthe Department of Urology, Odense University Hospital, and immediatelytransported to the Institute of Pathology. Each kidney, withor without tumor, was divided into cortex, outer medulla, andpapilla and frozen in liquid nitrogen. Tissue blocks were fixedin 4% paraformaldehyde overnight and embedded in paraffin. Othersections were embedded and frozen in CO2-cooled isopentane andkept at –80°C until use. In three kidneys, it waspossible to dissect a piece of the renal artery, which was immediatelyfrozen. Paraffin-embedded human tissue arrays and fetal kidneyswere obtained from the archive of the Department of Pathology,Odense University Hospital.
Human Umbilical Artery Endothelial Cell Culture
Primary cultures of human umbilical artery endothelial cellswere established by collagenase treatment of umbilical cords.The cords were obtained from the Department of Obstetrics andGynecology, Odense University Hospital, Denmark, after informedconsent was obtained. Human umbilical artery endothelial cellswere seeded on gelatin-coated tissue-culture plastic, and thecultures were maintained in EGM-2 medium (Clonetics, San Diego,CA) and kept at 37°C, 95% O2, 5% CO2. Cells were detachedwith trypsin-EDTA solution (Sigma, Rødovre, Denmark)and used until passage 7. Lipopolysaccharide (10 µg/ml,Sigma) was added to culture flasks for 5 h, and cell proteinwas isolated.
Molecular Protocols
Total RNA was isolated from tissue by columns (RNeasy Midi Kit,Qiagen, Albertslund, Denmark). RNA was quantified by measuringoptical density at 260 nM. RT-PCR was used to amplify sequencesspecific for human COX-1, COX-2, renin, and -actin (20). Allprimers were synthesized with 5' restriction sites for BamHI(sense) and EcoRI (antisense) (Invitrogen, Scotland) to allowfor directional cloning (vector pSP73, Promega-Ramcon, Birkerød,Denmark), sequencing, and in vitro transcription (20). Primerswere as follows: for COX-1, sense: 5'-ATG TCA TCA GGG AGT CTC-3',antisense: 5'-AAG CAG TCC AGG GTA GAA-3' (accession number xm011834,bases 1339 to 1514, 176 bp); and COX-2, sense: 5'-GTG AAA CCATGG TAG AAG-3', antisense: 5'-AGT AGT ACT GTG GGA TTG-3' (accessionnumber xm00173416, bases 1648 to 1924, 277 bp, with the 3' halfof the amplified sequence not found in COX-1); renin, sense:5'-ATG AAG AGG CTG ACA CTT-3', antisense: 5'-GAG AAA GCC ACTGAC TGT-3' (285 bp spanning two introns) (21); -actin, sense:5'-CCA AAG TTC ACA ATG TGG-3', antisense: 5'-CAC GAA AGC AATGCT ATC-3' (accession number xm004814, bases 1392 to 1579, 188bp).
Solution Hybridization and Ribonuclease Protection Assays
mRNA levels were estimated by solution hybridization followedby A/T1 ribonuclease protection assay as described (20). Protectedprobes were excised from dry gels and radioactivity was quantifiedin a counter. COX-1 and –2 probes had the same specificradioactivity, which permitted direct comparison of isoformmRNA levels.
Immunohistochemical and Immunofluorescence Analysis of Kidney Sections
Processing of tissue for immunohistochemical analysis was essentiallyas previously described (22). Two COX-2 antibodies were used;rabbit anti-human directed against amino-terminal amino acids50 to 111 (Santa Cruz, AH Diagnostics, Aarhus, Denmark), andrabbit anti-human against the carboxy-terminal amino acids 567to 599, unique to COX-2 (Cayman Chemicals Co., AH Diagnostics,Aarhus, Denmark). Both antibodies have been used previouslyon human tissue (7,9,11,23). COX-1 antibody was from Santa Cruz(goat-anti human). Antibodies directed against microsomal prostaglandinE synthase (PGES; rabbit anti-human), EP4 receptor (rabbit anti-human)and EP2 receptor (rabbit anti-rat) were from Cayman Chemicals.Antigen retrieval was carried out by microwave cooking in citratebuffer (Dako, Glostrup, Denmark) for 20 min or in a pressurecooker for 5 min. The sections were blocked with 5% dry milkin 0.05% Tween–Tris-buffered saline (TTBS), and then incubatedwith diluted primary antibodies overnight at 4°C. Next,the sections were incubated with horseradish peroxidase (HRP)-conjugatedsecondary antibody directed against the relevant species (Dako).The Santa Cruz COX-2 antibody was visualized according to thePower Vision poly HRP amplification kit (Immuno-vision TechnologyCo., Brisbane, CA). Signals were visualized by incubation for2 to 20 min with 0.01% diaminobenzidine (Dako) and 0.02% H2O2.For preabsorption controls, the antibody and 10 µg/mlpeptide was incubated in 5% milk-TTBS for 2 h. Immunolabelingfor EP4 receptor was carried out on unfixed cryosections thatwere incubated in 0.5% Triton-X–100 PBS, and next wereblocked in 5% dry milk–PBS. The sections were incubatedovernight at 4°C with EP4 primary antibody (1:50), and signalswere visualized as described above. Double-immunofluorescencelabeling for COX-1 and COX-2 was performed after blocking with5% milk-TTBS. Diluted primary antibodies were incubated overnightat 4°C, and the sections were then incubated with Alexa-fluor594 conjugated donkey–anti-goat antibody (1:250, MolecularProbes) followed by incubation with Alexa 488 conjugated goat–anti-rabbitantibody (1:200, Molecular Probes). Sections were inspectedwith an epifluorescence microscope (Olympus BX51).
Western Blot Test
Protein was isolated and quantitated as described (22). Twenty-microgramprotein samples were used for COX-2 assays and 60-µg samplesfor COX-1 assays. Proteins were separated by 4–12% SDS-PAGEand electroblotted (Bio-Rad Laboratories, Copenhagen, Denmark)onto PVDF Immobilon membranes (Millipore, Glostrup, Denmark).Membranes were blocked and incubated with anti–COX-2 antibodies(1:1000) or COX-1 antibody (1:1000) in 5% dry milk–TTBSovernight. Membranes were incubated with HRP-coupled secondaryantibody (1:2000) for 1 h. Bound secondary antibody was detectedby enhanced chemiluminescence kit (ECL plus Western BlottingDetection System, Amersham Biosciences, Horsholm, Denmark) andexposed to x-ray film. Proteins for PGES detection were separatedon a 10% SDS-PAGE gel and reacted with primary antibody (1:1000).
Statistical Analyses
All values are given as mean ± SEM. Unpaired t test wasused to determine statistical difference when two groups ofdata were compared. P < 0.05 was considered statisticallysignificant.
Distribution of COX mRNA and Proteins in Adult Human Kidney Regions
By ribonuclease protection, both COX transcripts were detectedin all regions of 11 adult human kidney samples that were selectedfrom patients who did not receive any chronic medication beforenephrectomy. COX-1 and –2 were expressed at equal levelsin kidney cortex, but COX-1 was more abundant than COX-2 inouter and inner medulla (Figure 1, A and B). There was a significantcortical-medullary gradient for both isoforms. Compared withcortex, COX-1 was expressed at 3 and 20 times higher levelsin outer and inner medulla, whereas COX-2 was modestly but significantlymore highly expressed in inner medulla compared with cortexand outer medulla (Figure 1B). Renin mRNA was detected in cortexonly, which confirmed correct separation of tissue (Figure 1A).The COX-2 antibodies, recognizing amino- and carboxy-terminalparts of human COX-2, reacted with a protein with the expectedsize of 72 kDa on Western blot tests of kidney cortex and innermedulla. Figure 1C shows the results with the COOH-terminalCOX-2 antibody. There was no significant difference in COX-2protein level between cortex and inner medulla. The COX-1 antibodydetected a single protein with the anticipated molecular sizeof 70 kDa. COX-1 protein level was significantly higher in theinner medulla compared with cortex, similar to the data on mRNAdistribution.
Figure 1. Distribution of cyclooxygenase (COX)-1 and COX-2 mRNA and protein in human kidney regions. (A) Autoradiographs depicting the distribution of COX-1, COX-2, and renin mRNAs in dissected human kidney regions from three patients. Messenger RNA determinations were performed in duplicate RNA samples with two amounts of total RNA from each human kidney region (20 and 40 µg from cortex and outer medulla and 10 and 20 µg from inner medulla). (B) Messenger RNA-cRNA hybrids were cut out of the dry gels and protected radioactivity in the probes was quantitated in a counter. Each column represents the average counts per microgram of RNA normalized with -actin mRNA level ± SEM from 11 separate kidneys. (C) Upper panels display the result of Western blot tests for COX enzymes with protein isolated from cortex and inner medulla of human kidneys. Twenty micrograms of protein from the cortex and inner medulla was used for the COX-2 assay, and 60 µg was used to detect COX-1. Band densities were evaluated; the lower panels display average densitometry units per square millimeter ± SEM (n = 3). OM, outer medulla; IM, inner medulla; * P < 0.05.
Immunohistochemical Localization of COX Isoforms in Adult and Fetal Human Kidney Sections
In adult kidney, COX-2 immunoreactivity was associated withpreglomerular, glomerular, and postglomerular vessels (Figure 2, a, b, d, f, and j, k).Preglomerular vessels of all calibersfrom arteries to arterioles were labeled in the smooth musclecell layer (Figure 2, b, d, and k; Figures 4A and 5B). In glomeruli,labeling was observed in parietal epithelial cells (Figure 2b)and in few intraglomerular cells, probably podocytes (Figure 4A)(8). In the postglomerular vasculature, labeling was associatedwith outer medullary vasa recta, as seen in the compound pictureof a longitudinal section and in cross sections of outer medulla(Figure 2, a, f, and j).
Figure 2. Immunohistochemical labeling of human kidney sections for cyclooxygenase (COX)-2 and COX-1. (a) Compound picture showing COX-2 immunoreactive protein in vessels of an outer medullary vasa recta bundle. Scale bar = 200 µm. In renal cortex, COX-2 was observed in vascular smooth muscle (b, d, k) and in the parietal glomerular epithelium (b), but not in the loop of Henle or the macula densa (b). (c) COX-1 was located in a few mesangial cells in glomerulus. Scale bar = 50 µm. Cross section of inner medulla revealed (g) COX-1 immunoreactivity in collecting ducts and (f) COX-2 in vasa recta and capillaries. Incubation of the primary antibodies with peptides used to raise the antibodies prevented labeling of tissue sections (h, i). Application of a separate COX-2 antibody directed against the amino terminal of COX-2 produced a similar staining result as with the carboxy-terminal antibody used in a, b, d, and f (j, k). Human fetal kidney displayed COX-2 in loop of Henle and macula densa (l) and in vasculature (m).
Figure 4. Immunofluorescence analysis of cyclooxygenase (COX) in human kidney and nonrenal tissues. (A) Human kidney cortex was double-labeled for COX-2 (green, a) and COX-1 (red, b), and colocalization was detected in few endothelial cells in the afferent arteriole (yellow, c). Scale bar = 50 µm. Cross section (d) and longitudinal section (e) of human kidney outer medulla and inner medulla (f) labeled for COX-1 (red) and COX-2 (green), showing separate localization. Scale bar in d and e = 200 µm; f, 50 µm. (B) Labeling of human fetal kidney tissue at gestational week 30 for COX-2. COX-2 signals were associated with glomeruli (g), loops of Henle (g) arteries and arterioles (h), and vasa recta (i). The glomeruli are small compared with (A). Scale bar = 50 µm. (C) Labeling of a human tissue array for COX-2 (green fluorochrome) showed a vascular localization: skeletal muscle (j), bladder (k), spleen (l), ventricle (m), gallbladder (n), and small intestine (o). Scale bar = 50 µm.
Figure 5. Immunolocalization of potential targets for cyclooxygenase (COX)-derived prostanoids in human kidney. (A) Localization of microsomal prostaglandin E2 synthase (PGES) immunoreactive protein in human kidney. In cortex, PGES was consistently associated with the macula densa (a through c) and with collecting ducts (d). Scale bar = 50 µm. In medulla, PGES was observed uniformly in collecting duct epithelium (e). Anti-PGES antibody detected a single protein with the expected size by Western blot test with protein from human kidney inner medulla (f). (B) Localization of prostaglandin E2 (PGE2) EP4 receptors in human kidney. In adjacent serial sections, COX-2 and EP4 receptors were colocalized in arteries (g, h) and preglomerular arterioles (i, j). Scale bar in (g) = 50 µm. (C) Localization of cAMP-coupled PGE2-EP2 receptor immunoreactive protein in human kidney. EP2 was associated with distal convoluted tubule (k) and loop of Henle (l). Arrows indicate the macula densa region where the EP2 signal waned (k).
The inner medullary capillary network was labeled for COX-2,which implies that COX-2 is expressed in endothelium (Figure 2, f and j).The immunopositive vessels were identified on thebasis of the presence of erythrocytes in their lumen. In adultkidney, no COX-2 immunoreactivity was associated with collectingducts, proximal tubules, loops of Henle, distal tubules, ormedullary interstitium (Figure 2, a, f, and j). Notably, wedid not observe immunolabeling in macula densa in any of thesamples analyzed (Figure 2b). A second, COX-2–specificantibody directed against the NH2-terminal of human COX-2 wasapplied. The same pattern of labeling was obtained with thisantibody (Figure 2, j and k). In two human fetal kidneys (gestationalweeks 29 and 30), COX-2 immunoreactivity was associated withthe parietal epithelial cells of Bowman’s capsule andwith endothelium and vascular smooth muscle (Figure 2, l and m;Figure 4B). COX-2 was not found in the nephrogenic zone,but was associated with loops of Henle, including macula densacells of mature juxtamedullary glomeruli (Figures 2l and 4B),as previously reported (8–10).
In adult kidney, COX-1 immunoreactivity was associated withendothelial cells of arteries, with mesangial cells at the glomerularhilum and with cortical interstitial cells (Figure 2, c, e, and g).COX-1 immunoreactivity was detected in collecting ductprincipal cells in cortex and medulla (Figure 2, c and e). Omissionof primary COX antibodies or preabsorption of antibodies withsurplus of the respective peptides prevented labeling (Figure 2, h and i).
Vascular Expression of COX Enzymes
Vascular expression of COX isoforms was examined by Westernblot tests of proteins isolated from human renal artery (n =2). Both COX-2 antibodies, directed against amino- and carboxy-terminalepitopes, reacted with a protein from renal artery that wasmore abundant compared with renal inner medulla and had theexpected size of COX-2 (Figure 3, A and B). Significant COX-1expression was also detected in renal artery but was less abundantcompared with COX-1 in inner medulla (Figure 3C). Cultured humanendothelial cells exhibited COX-2 signals when analyzed on Westernblot test (Figure 3D)
Figure 3. Western blot tests for cyclooxygenase (COX)-2 using protein isolated from human kidney inner medulla (IM) and human renal artery. A total of 20 µg was used for the COX-2 assay, and 60 µg was used for the COX-1 assay. (A) COX-2 antibody directed against an epitope in the carboxy-terminal domain of COX-2. (B) COX-2 antibody directed against an epitope in the amino-terminal of human COX-2. (C) COX-1 antibody. (D) Western blot tests for COX-2 with 20 µg protein isolated from cultured human umbilical artery endothelial cells that were incubated with or without lipopolysaccharide (LPS, 10 µg/ml) for 5 h. Twenty micrograms of protein from IM was applied as positive control.
Cellular Localization of COX-2 in Human Renal and Extrarenal Vasculature
Human kidney sections were double-labeled by immunofluorescencefor COX-1 and –2. Figure 4A, a through c, shows an afferentarteriole where COX-2 immunofluorescence was associated withsmooth muscle and endothelial cells and with parietal epithelialcells of Bowman’s capsule (a, green). The COX-1 antibodylabeled endothelial cells in the afferent arteriole (b) andmesangial cells in the glomerulus (b). Endothelial cells werethe only cells that occasionally exhibited both COX-1 and –2signals (c, arrow). In renal outer medulla, COX-2 fluorescencewas restricted to vasa recta (d, e, green), whereas COX-1 wasuniformly associated with collecting duct epithelium (d, e,red). In the inner medulla, COX isoforms were associated withsimilar structures as in outer medulla; COX-2 was observed inmedullary capillaries (f, green). In fetal human kidney tissueat gestational week 30 (Figure 4B), COX-2 signals in cortexwere associated with glomeruli (g), loops of Henle (g), andarteries (h). In the immature medulla, vasa recta were COX-2positive (i).
Next, it was addressed whether vascular expression of COX-2was specific for the kidney. An array of adult human organswas labeled with the NH2-terminal COX-2 antibody (Figure 4C).Significant fluorescence signals were associated with submucosalarteries and arterioles in ventricle (m), gallbladder (n), jejunum(o), and urinary bladder (k). Arterial and arteriolar smoothmuscle was labeled in skeletal muscle (j) and spleen (l) andin dermis, lung, and placenta (not shown).
Localization of Downstream Targets for COX Products in Human Kidney
Microsomal PGES is responsible for conversion of the COX productprostaglandin H2 to prostaglandin E2 (PGE2). PGES immunoreactivityin cortex was associated with the macula densa and not observedin adjacent epithelial cells (Figure 5A, a through c). PGESlabeling was detected in a subset of cortical collecting ductcells and in all inner medullary collecting duct cells (Figure 5A, d and e).On Western blot test, the PGES antibody detecteda single protein with the expected size of 16 kDa (Figure 5A, f).
Next, Gs-coupled vasodilator receptors for PGE2, EP2 and EP4,were localized in human kidney. EP4 labeling was detected invascular smooth muscle cells of large (Figure 5B, h) and smallerafferent (Figure 5B, j) glomerular vessels (24). In adjacentserial sections, COX-2 was associated with the same smooth musclecells as EP4 (Figure 5B, g and i). EP4 immunoreactivity wasdetected in vasa recta bundles (not shown). Immunopositivityfor the EP2 receptor was associated with thin descending andascending limbs of Henle’s loop (Figure 5C, l) (25). EP2immunolabeling was detected in the post–macula densa distalconvoluted tubule and in connecting tubule (Figure 5C, k). EP2receptor labeling was associated predominantly with the basolateralaspect of the epithelial cells. EP2 immunoreactivity was notobserved in any part of the renal vasculature or in the maculadensa (Figure 5C, k, arrows).
Effect of Renal Artery Stenosis, Angiotensin II (AngII) Inhibitors, and COX Inhibitors on COX Expression in Human Kidney
COX expression was analyzed in kidneys with renal artery stenosis;in kidneys from patients who had preoperatively received angiotensin-convertingenzyme (ACE) inhibitors, or AT1 receptor antagonists; and inpatients who had received acetylsalicylic acid. COX-2 mRNA abundancewas significantly higher in cortex and medulla from kidneyswith arterial stenosis compared with control kidneys (Figure 6, A through C).Renin expression was strongly elevated in threeof the four kidney cortices with arterial stenosis comparedwith six controls (Figure 6A). COX-1 and COX-2 expression wasnot changed in kidneys from patients who had received AT1 receptorblockers, ACE inhibitors, or acetylsalicylic acid (not shown).Kidneys with artery stenosis exhibited tubular atrophy and interstitialfibrosis. The vasculature displayed medial and intimal thickeningand some arteriolar hyalinosis. COX-2 immunoreactivity was associatedwith vascular smooth muscle in the thickened media layer. Ingeneral, there was a poor morphologic resolution in the corticallabyrinth because of the end-stage nature of the kidneys. Therefore,it was not possible to identify the macula densa or any otherspecific tubular segment in these kidneys.
Figure 6. Expression of cyclooxygenase (COX) in kidneys with renal artery stenosis. (A, B) Autoradiographs displaying hybrids for COX-1, COX-2, and renin mRNAs in human kidney regions from separate patients with renal artery stenosis and renovascular hypertension (RVH). tRNA is a negative control where probes hybridized with yeast tRNA, and probe bands display the radiolabeled probe with no tRNA. (C) Hybrids were cut from the gels, and protected radioactivity in the probes was assayed in a counter. Each column displays average, actin-normalized, COX-2 mRNA level ± SEM (RVH, n = 4; control cortex, n = 6, and medulla, n = 5). OM, outer medulla. (D) COX-2 immunostaining of kidney tissue from a patient with renal artery stenosis. Abnormal cortical architecture is seen with sclerotic glomeruli, obliterated vessels, fibrosis, and loss of tubules. COX-2 was detected in vessel media and endothelium in cortex. Inner medulla was atrophied in these kidneys. Scale bar = 50 µg.
In the present investigation, the major finding is that cyclooxygenase(COX-1 and COX-2) was expressed constitutively in all regionsof adult human kidney with distinct cellular localization. COX-1was associated with collecting ducts. COX-2 was associated withmedia smooth muscle and vascular pericytes of all segments ofthe pre- and postglomerular vasculature and was significantlyelevated in kidneys from patients with renal artery stenosis.In fetal kidneys, COX-2 was expressed in vasculature and glomeruli,but also in loop of Henle and macula densa cells. Thus, COX-2was detected consistently in tubular epithelium only in fetalkidneys, whereas vascular expression was a constant featureof human kidney established during fetal life.
Because of the reported controversies on COX-2 localizationin human kidney, the specificity of the COX-2 antibody was ofmajor concern. Two separate anti-human COX-2 antibodies directedagainst different, specific epitopes yielded a similar patternof histochemical labeling that was different from that of COX-1.Labeling was prevented by preabsorption with the peptides usedfor immunization. Both antibodies reacted with a protein withthe expected molecular size of COX-2 on Western blot test ofkidney tissue and renal artery, and the intrarenal distributionof COX-2 protein corresponded to COX-2 mRNA. Moreover, COX-2immunopositive vessels were encountered in all kidney zonessimilar to COX-2 protein and mRNA. COX-2 has previously beenobserved in adult human kidney vasculature with a differentCOX-2 antibody (8) and also in nonrenal human arteries (23)and in vascular endothelium of several other species (26,27).
The vascular localization of COX-2 was not restricted to humankidney because an array of human organs displayed immunolabelingassociated with media of arteries and arterioles. Together,the data indicate that renovascular expression of COX-2 is ageneral feature not causally related to the presence of adjacentmalignant cells in the human kidney tissue analyzed. Moreover,despite different gender (three women, eight men), age (range,45 to 83 yr, median 63 yr), tumor size, and histopathologicaltumor type (clear cell, transitional papillary tumor), therewas very little variation between patients in intrarenal COXmRNA and protein levels. This suggests constitutive expressionin adult human kidney. It is established that vascular COX-2expression is stimulated by the shear stress-nitric oxide pathway,by AngII, and by inflammatory mediators and cytokines (28–33).COX-2 couples preferentially with prostacyclin (PGI) synthaseand inducible PGE2 synthase (34), and COX-2 is the predominantsource of PGI2in vivo (2,23,35,36). Tonical vascular prostacyclinsynthesis is crucial to antagonize thromboxane-mediated, injury-inducedproliferation and thrombosis (37). The present observation ofwidespread vascular COX-2 offers an explanation for the inhibitoryeffect of COX-2 antagonists on prostacyclin synthesis in humans(2,23,35,36).
In kidneys with arterial stenosis, vascular COX-2 expressionwas augmented in cortex and medulla. Human kidneys with arterialstenoses release net prostaglandin to the systemic circulationand COX blockers cause a drop of GFR (19), plasma renin, andBP (17–19,38). Thus, vascular COX-2 activity, either fromsmooth muscle or from endothelium, may contribute to elevatedrenal prostaglandin synthesis under conditions with diminishedblood flow to the kidney. PGE2 and/or PGI2 may serve to supportvessel patency and blood supply to the ischemic kidney throughinteraction with vascular IP or EP4 receptors (present data)(24,39). AngII has been shown to induce COX-2 in smooth muscle(29), but COX-2 mRNA was not changed in nephrectomy samplesfrom patients that received AngII receptor blockers or ACE inhibitors.However, because of the few samples analyzed, we cannot excludea regulatory role of AngII on COX-2 in the human kidney.
In cortex, immunolabeling for PGES was associated with maculadensa and collecting ducts and was not observed in the vasculaturesimilar to rat (40). We did not observe any COX isoform associatedwith the macula densa in the adult kidneys analyzed. This observationessentially confirms previous reports where COX-2 was absentin normal adult human kidney macula densa (7,8,11) but presentin fetal kidney (9). COX-2 is thought to be induced in humanmacula densa in conditions with low physiologic, or compromised,transepithelial NaCl transport (9,10,11,13). Dependence of plasmarenin on COX-2 has been shown in normal volunteers given a low-saltdiet or furosemide (16) and in patients with Bartter syndrome(15). The data suggest that COX-2–mediated prostaglandinsynthesis contributes to macula densa–mediated signaltransfer to renin-producing granular cells in these situations.None of the patients in the study presented here had overt heartdisease or received loop diuretics; all were elderly and presumablyNaCl replete. Thus, we propose that COX-2 protein is below thedetection limit in macula densa of sodium-replete humans butthat it can be rapidly induced and generate substrate for PGESin states of low NaCl intake, diuretic treatment, or defecttransport proteins.
In summary, the present data show constitutive expression ofboth COX isoforms in normal adult human kidney. COX-1 was colocalizedwith PGES in collecting ducts. COX-2 was localized in vascularsmooth muscle and endothelium together with EP4 receptors bothin kidney and extrarenal tissues. COX-2 was expressed in cTALand macula densa in human fetal kidney during and after activenephrogenesis.
Acknowledgments
This work was supported by grants from the Danish Medical ResearchCouncil (22010122, 22010159), the Novo Nordisk Foundation, theDanish Heart Foundation (01123022896), the Danish Medical AssociationResearch Fund, A. J. Andersens Foundation, J. C. ChristoffersensFoundation, and Johannes Fogs Foundation. The technical assistanceof Mette Fredenslund and Inge Andersen is gratefully acknowledged.We thank Anthony M. Carter for English-language revision.
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Received for publication December 10, 2003.
Accepted for publication February 5, 2004.
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Top
Abstract
Introduction
-actin (20). All primers were synthesized with 5' restriction sites for BamHI (sense) and EcoRI (antisense) (Invitrogen, Scotland) to allow for directional cloning (vector pSP73, Promega-Ramcon, Birkerød, Denmark), sequencing, and in vitro transcription (20). Primers were as follows: for COX-1, sense: 5'-ATG TCA TCA GGG AGT CTC-3', antisense: 5'-AAG CAG TCC AGG GTA GAA-3' (accession number xm011834, bases 1339 to 1514, 176 bp); and COX-2, sense: 5'-GTG AAA CCA TGG TAG AAG-3', antisense: 5'-AGT AGT ACT GTG GGA TTG-3' (accession number xm00173416, bases 1648 to 1924, 277 bp, with the 3' half of the amplified sequence not found in COX-1); renin, sense: 5'-ATG AAG AGG CTG ACA CTT-3', antisense: 5'-GAG AAA GCC ACT GAC TGT-3' (285 bp spanning two introns) (21); 
). In glomeruli, labeling was observed in parietal epithelial cells (Figure 2b) and in few intraglomerular cells, probably podocytes (Figure 4A) (8). In the postglomerular vasculature, labeling was associated with outer medullary vasa recta, as seen in the compound picture of a longitudinal section and in cross sections of outer medulla (Figure 2, a, f, and j). 
), as previously reported (8–10). 
65 years of age with systemic hypertension and osteoarthritis. Am J Cardiol 90: 959–963, 2002[CrossRef][Medline]
