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作者：Valentina Câmpean, Franziska Theilig, Alex Paliege, Matthew Breyer, and Sebastian Bachmann作者单位：1 Anatomisches Institut, Charité, Humboldt Universität, D-10098 Berlin, Germany; and 2 Division of Nephrology, Vanderbilt University, Nashville, Tennessee 37232-2372 ; L2 Q) |: \9 t$ x/ L# Q' ~7 U" e
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/ P3 Q8 z9 E; L! p3 [ 【摘要】
9 z" W" } W) s Prostanoids derived from endogenous cylooxygenase (COX)-mediated arachidonic acid metabolism play important roles in the maintenance of renal blood flow and salt and water homeostasis. The relative importance of COX-1 and COX-2 isoforms is under active investigation. We have performed a comprehensive histochemical analysis by comparing rat and mouse kidneys for cellular and subcellular localization of COX-1 and -2 and microsomal-type PGE synthase (PGES), the rate-limiting biosynthetic enzyme in PGE 2 synthesis. A choice of different sera was compared, and the results were confirmed by antigen-retrieval techniques, in situ hybridization, RT-PCR, and the use of COX knockout mice. In the glomerulus, significant COX-1 expression was detected in a subset of mesangial cells. Along the renal tubule, the known COX-2 expression in cTAL and macula densa was paralleled by PGES staining. In the terminal distal convoluted tubule, connecting tubule, and cortical and medullary collecting ducts, a significant COX-1 signal was colocalized with PGES; COX-2 was not found in these sites. Intercalated cells were generally negative. Cortical fibroblasts were COX-1 and PGES positive in mice, whereas in rats only PGES could be reliably detected. Lipid-laden interstitial cells of the inner medulla were COX-1, -2, and PGES positive. Vascular smooth muscle cells were not stained. The present data support prominent functions of renal prostanoids, predominantly PGE 2, by defining expression sites of the key enzymes for their biosynthesis in the rat and mouse. Results define the renal cell types involved in prostaglandin autacoid functions within spatially restricted sites such as the juxtaglomerular apparatus, mesangium, distal convolutions and collecting duct, and in compartments of the renal interstitium. / H8 t- w" K' N( b
【关键词】 mesangium thick ascending limb distal convolutions collecting duct interstitium. K2 |" B# a( {: c/ F# H B
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THE MAMMALIAN KIDNEY IS A major site for the synthesis and action of products derived from cyclooxygenase [COX; prostaglandin synthase (PGES); G 2 /H 2 ]. The enzymatic conversion of arachidonate to PGH 2 is followed by further metabolism into several bioactive prostaglandins such as PGE 2, the major renal prostaglandin, and others like PGI 2, PGF 2, PGD 2, or thromboxane A2 (TxA 2; for a review, see Ref. 43 ). PGE 2 is preferentially catalyzed by the rate-limiting terminal prostanoid synthase [PGE 2 synthase (i.e., PGES)] ( 15, 24 ).- c/ c! F: u5 `- j
/ f- h1 l, M, d. V) VAs elsewhere in the organism, prostaglandins play multifold roles in the physiology and disease of the kidney. They interfere importantly with the modulation of local hemodynamics, renin release, and tubular salt and water reabsorption (for a review, see Refs. 19 and 40 ). Renal medullary prostaglandins are involved in the pathogenesis of hypertension, maintenance of renal blood flow, and promotion of urinary salt excretion ( 37 ). They further play an important role in electrolyte disorder syndromes such as the antenatal Bartter syndrome ( 35 ) and in inflammatory renal disease (61). Renal prostaglandins act as paracrine autacoids, causing local, specific effects according to the renal zonation and distribution of cell types ( 37, 48, 49 ); this is also reflected by the spatial distribution of the various receptor subtypes (for a review, see Ref. 6 ).
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, {7 f& d, g3 lThere are two COX isoforms that have been identified recently by molecular approaches. Both exhibit similar biochemical activity in converting arachidonate to PGH 2 ( 9 ) and are similar in molecular mass (70 and 71 kDa), but they differ largely in amino acid sequence and also with respect to the availability of the downstream-acting prostanoid synthases and the prostanoids ultimately formed ( 43 ). They also show differential distribution and regulation. COX-1, the constitutive form, is widely expressed in the organism and is considered to have "housekeeping" functions providing the majority of prostaglandin production ( 9 ). The inducible isoform, COX-2, may be increased during inflammation, but among few other tissues it is also expressed constitutively in topographically restricted sites of the kidney ( 17 ). The distinct role of the latter has also been elucidated by the use of mutant mice; the kidneys of COX-2-deficient mice fail to develop normally ( 33 ), whereas those lacking the gene for COX-1 production do not show conspicuous symptoms under normal conditions ( 1 ). Isoform-specific inhibitors have recently attracted a great deal of interest because the selective targeting of pathophysiological sources of prostaglandins is considered a promising means of avoiding any adverse renal effects during pharmacological treatment (for a review, see Ref. 18 ).
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Localization of the renal sources of prostaglandin synthesis and release is thus a major issue that has been studied in a number of mammalian species under constitutive and regulated conditions ( 15, 19, 20, 23, 29, 31, 41, 42, 45, 46, 49, 51 ). However, important aspects of tissue localization have remained unresolved or controversial, owing to conventional problems in the interpretation of histochemical staining procedures. In particular, COX expression in glomerular cells ( 10, 30, 31 ), collecting duct cell types ( 11, 42, 47 ), and in the cortical vs. medullary interstitium ( 31, 37, 50 ) has been reported inconsistently, and a segmental assignment of cortical COX-1 immunoreactivity has been lacking. The availability of new antibodies and intensified histochemical detection protocols have prompted us to comparatively evaluate in detail COX-1, COX-2, and microsomal PGES distribution using different morphological techniques. Particular emphasis has been put on the identification of epithelial expression sites in the nephron and collecting duct system. With regard to the increasing interest in mutant mice, this study has been comparatively performed in normal rats and mice. Mutant mice have been used as controls for signal specificity. In the present study, we thus aimed to present a comprehensive histochemical analysis to support functional interpretation of site-specific prostaglandin synthesis.1 O- J; E: U- d- w& ]2 P
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2 i2 e; P, I: ^Animal preparation. Adult male Sprague-Dawley rats (200-g body wt) were obtained from the local animal facility. Adult male COX-1 and COX-2 null mutant knockout mice (COX-1 -/- and COX-2 -/- ) and wild-type control mice (COX-1 / ) were from a mouse colony at Vanderbilt University and at the National Institutes of Health (NIH; kindly provided by J. B. Schnermann). Information on the genetic background of these mice can be found elsewhere ( 37 ). All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication 85-23, Revised 1996) and the German Law for the Protection of Animals. The kidneys were perfused retrograde through the abdominal aorta using PBS adjusted to 330 mosmol/kgH 2 O with sucrose, pH 7.4, for 20 s as described before ( 38 ). Next, a solution of 3% paraformaldehyde in PBS was infused for 5 min, followed by PBS adjusted to 330 mosmol/kgH 2 O for 1 min. For cryostat sectioning, tissues were protected from freezing artifacts by subsequent overnight immersion in PBS adjusted to 800 mosmol/kgH 2 O by sucrose (pH 7.3), shock-frozen in liquid nitrogen-cooled isopentane, and stored at -70°C. For preembedding immunohistochemical analysis, tissue blocks were postfixed for 12 h in 3% paraformaldehyde in PBS containing 0.05% glutaraldehyde, rinsed, and subsequently stored in PBS. For the paraffin technique, tissue was postfixed in 3% paraformaldehyde for another 12 h and then dehydrated and standard paraffin embedded.* R- v- M1 P+ c5 y3 | k
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Light microscopic immunohistochemistry. Five-micrometer-thick cryostat and paraffin sections were used. Paraffin sections were dewaxed and rehydrated, heated in a microwave oven in 2.9 g/l citrate buffer (pH 6.0) for 20 min, or incubated in a pressure cooker for 90 s in preheated target retrieval solution from a catalyzed signal amplification kit (DAKO, Hamburg, Germany), producing a streptavidin-biotin-peroxidase signal reaction. After being blocked with 5% skim milk in PBS (pH 7.4), sections were incubated with primary antibody for 2 h at room temperature and then overnight at 4°C. In double-labeling experiments, suitable secondary antibodies coupled to different fluorochromes were applied (Dianova, Hamburg, Germany).
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Enzymes involved in prostaglandin synthesis were identified using the following antibodies: rabbit polyclonal antibody against amino acids 274-288 of COX-1 (dilution 1:7,000; batch 160109; Cayman Chemical, Ann Arbor, MI); rabbit polyclonal antibody against an NH 2 -terminal peptide of COX-1 (amino acids 63-124; dilution 1:500; batch 1754; Santa Cruz Biotechnology, Heidelberg, Germany); rabbit polyclonal antibody against amino acids 584-598 of COX-2 (dilution 1:500; batch 160126; Cayman Chemical); goat polyclonal antibody against a COOH-terminal COX-2 peptide (dilution 1:500; batch 1747; Santa Cruz Biotechnology); and rabbit polyclonal antibody against an NH 2 -terminal peptide of microsomal type PGES (amino acids 59-75; dilution 1:5,000; batch 160630; Cayman Chemical). Nephron portions were identified by double labeling with segment-specific antisera on the same sections or on consecutive sections depending on the origin of the antibody. Rabbit antibody to Tamm-Horsfall protein (gift from J. Hoyer, Philadelphia, PA); rabbit antibody to the Na-Cl cotransporter [NCC; gift from David H. Ellison, Portland, OR ( 38 )]; mouse monoclonal antibody against calbindin D 28K (Sigma, Taufkirchen, Germany); and guinea pig antiserum against the basolateral Na/Ca exchanger [kindly provided by R. Reilly, New Haven, CT ( 38 )] were used. To confirm the specificity of the antibodies, control experiments were performed by replacing primary antibodies with 5% skim milk in PBS or by preabsorption with the respective peptides used for immunization as far as they were commercially available.
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Slides were evaluated with a digital Spot camera from Diagnostic Instruments, processed with Meta Vue software from Universal Imaging supported by Visitron System (Puchheim, Germany), and viewed with a Leica DMRB light microscope equipped with interference contrast optics and an HBO fluorescence lamp.# u! \$ l7 L1 d5 E3 ~8 ~
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Ultrastructural analysis. For preembedding immunoperoxidase labeling, 40-µm-thick kidney slices generated with a Vibratome (Leica, Weiterstadt, Germany) were incubated overnight in microtiter plates with the appropriate COX antibodies at relatively high concentrations (dilutions 1:10-1:200), postfixed with 1% osmium tetroxide, stained en bloc with uranyl acetate, and flat embedded in Epon 812. Ultrathin sections were analyzed after additional contrasting in uranyl acetate and lead citrate. Sections were examined with a Leo 906 electron microscope (Zeiss, Oberkochen, Germany). Semithin sections were evaluated as well.
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6 A& ?0 O0 ?9 H7 P% a# @3 oIsolation of glomeruli and RT-PCR analysis. Glomeruli were isolated as described previously ( 32 ). Total RNA from isolated glomeruli was extracted using the guanidinium thiocyanate method. Five micrograms of total RNA from each sample were reverse transcribed. The following sets of oligonucleotide primers were used to amplify cDNA: 5'-TATCCGTTGTGGATCTGAC-3' and 5'-TGGTCCAGGGGTTTCTTAC-3' (GAPDH; 300 bp); 5'-CCTTCCGTGTGCCAGATTAC-3' and 5'-GGCTGGCCTAGAACTCACTG-3' (COX-1; 475 bp); 5'-ACACTCTATCACTGGCATCC-3' and 5'-GAAGGGACACCCTTTCACAT-3' (COX-2; 585 bp); and 5'-TGTACGCGGTGGCTGTCATC-3' and 5'-GCCAGAACATAGGCCCCGG-3' (PGES; 300 bp). Amplification was performed using Taq polymerase for 24-35 cycles with an automated thermal cycler (PerkinElmer, Weiterstadt, Germany). Each cycle consisted of the following steps: denaturation at 98°C, annealing at 63°C, and extension at 72°C. The amplified DNA fragments had the expected lengths as validated by agarose gel electrophoresis and ethidium bromide staining.
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1 H M- y1 G# F4 Q& l; }Western blot analysis. Freshly isolated kidneys from rats and mice were dissected and cut into small pieces. The samples were homogenized in ice-cold buffer containing 1% Triton X-100, 0.2% SDS, and 1 tablet of protease inhibitor cocktail (Roche, Grenzach-Wyhlen, Germany) in 25 ml PBS buffer, followed by a 10-min treatment in an ultrasonic bath. The homogenates were centrifuged (14,000 g at 4°C), and the supernatants were stored at -80°C until assayed. Samples (100 µg/lane) were run on 10% gradient polyacrylamide minigels and blotted onto nitrocellulose membranes. Blots were blocked overnight with 5% nonfat skim milk and then incubated for 1 h at room temperature with antibody against COX-1, COX-2, or PGES. After extensive washing with 5% Tween in PBS, blots were incubated with horseradish peroxidase-coupled IgG. A signal was developed according to the manufacturer's protocol (Sigma).
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3 k F1 Z$ z1 `8 C, b- O* dIn situ hybridization. Digoxigenin(DIG)-UTP-labeled riboprobes and an alkaline phosphatase-dependent signal were generated as described ( 2 ). COX-1 riboprobe was made from an 800-bp partial cDNA fragment of rat COX-1, COX-2 riboprobe from a 1,300-bp partial cDNA fragment of rat COX-2, and NCC riboprobe from a 700-bp partial cDNA fragment of rat NCC. COX-2 and NCC fragments were subcloned into the Eco RV site of pBluescript vector (Stratagene, La Jolla, CA) and the COX-1 fragment into the pCR II-TOPO vector (In-Vitro Gen, Giessen, Germany). To generate sense and antisense riboprobes, the vectors were linearized by Xho I/ Bam HI for COX-1, Kpn I/ Xho I for COX-2, and Xho I/ Sac I for NCC (Roche, Mannheim, Germany), respectively. RNA probes were synthesized and in vitro transcribed in the presence of DIG-UTP and T3, T7, or SP6 polymerases (Roche). For in situ hybridization, 7-µm-thick paraffin sections were treated according to an established protocol ( 2, 38 ). Riboprobes were applied at concentrations between 10 and 15 ng/ml, and hybridization was performed at 40-50°C. For control, sense probes were applied in parallel with antisense probes. Sheep anti-DIG-alkaline phosphatase conjugate (DAKO) was diluted 1:50 in blocking medium. A signal was generated using 4-nitro blue tetrazolium chloride, 5-bromo-4-chloro-3-indolylphosphate, and levamisole dissolved in 0.1 M Tris · HCl, 0.1 M NaCl, and 0.05 M MgCl 2, pH 9.5. Slides were rinsed with PBS and placed under a coverslip with PBS-glycerol and viewed using brightfield microscopy as described above. Radiolabeled in situ hybridization was performed according to established protocols. Before hybridization, tissue sections were deparaffinized, refixed in paraformaldehyde, treated with proteinase K (20 µg/ml), washed with PBS, refixed in 4% paraformaldehyde, and treated with triethanolamine plus acetic anhydride (0.25% vol/vol). The respective 35 S-labeled antisense and sense riboprobes were hybridized to the section at 55°C for 18 h. After hybridization and stringency washes, sections were treated with RNase A (10 µg/ml) at 37°C for 30 min. Slides were dehydrated, air-dried, dipped in photoemulsion (Ilford K5; Knutsford, Cheshire, UK), and exposed for 4-5 days at 4°C. Photomicrographs of the radiolabeled in situ hybridization were viewed with darkfield optics using a Zeiss photomicoscope equipped with a digital camera (Spot-Cam; Diagnostic Instruments, Sterling Heights, MI).
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+ a9 Z9 x3 q$ G' P8 o FAntibodies against COX isoforms. A choice of commercially produced antisera against COX-1, COX-2, and PGES is presently available; however, the present experience has shown that detailed examination of cross-reactivity was in fact necessary to assess the specificity of the immunological probes. Some of the antibodies tested showed significant cross-reactivity among isoforms, contrary to product specifications. Different antibodies to the same epitope did not necessarily yield the same immunohistochemical staining patterns, whereas others stained structures unlikely to express COX isoforms. Isoform specificity, as outlined by the producer, was not always given because sera labeled anti-COX-1 were raised against peptides with similarities to COX-2. Therefore, only comparative analysis of several different approaches such as conventional immunofluorescence staining protocols, ultrastructural immunoperoxidase labeling, and in situ hybridization was successful. In light microscopy, the use of fluorescent antibody labeling resulted in very clear-cut staining with no major background problems; however, brightfield microscopy with peroxidase staining using an antigen-retrieval kit was clearly more sensitive, so that weak signals such as epithelial COX-1 that were not well detected by immunofluorescence could be detected satisfactorily with this technique. The use of controls including COX-1 and COX-2 knockout mice was essential for confirmation of these results. The best signals were obtained using rabbit polyclonal antibody to murine COX-1 (batch 160109; Cayman Chemical); goat polyclonal anti COX-2 antibody (batch 1747; Santa Cruz Biotechnology); and rabbit polyclonal antibody to microsomal PGES (batch 160630; Cayman Chemical).
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Identification of kidney structures. For a precise assignment of prostaglandin synthesis to identified portions of the renal tubule, we used morphological criteria as well as the segmental coexpression of defined markers ( 3, 6 ). Glomerular and juxtaglomerular cells were identified by fine structural criteria. The proximal tubule was recognized by the presence of brush border; thin limbs and vasa recta by their location within the vascular bundles; medullary and cortical thick ascending limb (mTAL and cTAL, respectively) by the presence, and macula densa by the absence, of Tamm-Horsfall protein; distal convoluted tubule (DCT) by the presence of the NCC; and parts of the DCT and the connecting tubule (CNT) by the Na/Ca exchanger and calbindin. Collecting ducts were identified by their microanatomic location, epithelial height, and presence of intercalated cells; and interstitial cells were identified based on their shape, location, and immunoreactive pattern.
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Cortex. The glomerulus and juxtaglomerular apparatus revealed significant COX-1 immunoreactivity in the extraglomerular mesangium in both species. In addition, a subset of mesangial cells of the glomerular tuft was markedly COX-1 positive ( Fig. 1 ). Ultrastructural distribution of COX-1 in rat mesangial cells showed a mild cytoplasmic and a stronger nuclear envelope staining ( Fig. 1 a ). Along the nephron, COX-1 immunoreactivity was absent from thin limbs of the loop of Henle, mTAL, cTAL, macula densa, postmacular segment, and initial DCT ( subsegment 1 or DCT1; Fig. 2, a and b ); the onset of a COX-1-immunoreactive signal was detected in terminal DCT, CNT, and cortical collecting duct (CCD) principal cells, as revealed by immunofluorescence staining ( Fig. 2, c - l ) and by an antigen-retrieval technique using CSA-immunoperoxidase staining ( Fig. 3 ). Both techniques showed largely the same results in rats and mice. Intercalated cells were negative. In segments from COX-1 knockout mice, no signal was detected ( Fig. 3, g and h ). Fine structural distribution of epithelial COX-1 immunoreactivity similar to that in mesangial cells showed a moderate cytosolic staining and an enhanced signal at the perinuclear envelope, as revealed by preembedding immunoperoxidase staining ( Fig. 3, c and d ). In mice, a major proportion of the cortical interstitium showed COX-1-immunoreactive interstitial fibroblasts in the cortical labyrinth ( Fig. 4 a ). In situ hybridization as well revealed the presence of COX-1 transcript in the distal convolutions of rat kidney ( Fig. 5, a and b ); in mice, COX-1 mRNA distribution was analogous but weaker than in rats, with only a few scattered cells showing significant staining ( Fig. 5, c and d ). Arteriolar endothelia only occasionally showed mildly positive COX-1-immunoreactive staining in both species.
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) D, s9 e- I- kFig. 1. Distribution of cyclooxygenase (COX)-1 immunoreactivity (IR) in glomerulus and juxtaglomerular apparatus. a : Ultrastructural immunoperoxidase staining reveals COX-1 IR in a single mesangial cell; note the perinuclear concentration of signal. b - d : In rats and wild-type mice (w.t.), extraglomerular mesangial cells stain significantly for COX-1, whereas in COX-1 knockout mice (k.o.) there is no signal. Arrowheads, macula densa; G, glomerulus. Ultrathin section ( a ) and paraffin sections, hematoxylin stain, and interference contrast ( b - d, respectively) are shown. Approximate magnifications: x 5,500 ( a ), x 800 ( b and c ), and x 400 ( d ).1 X* M8 D, S/ U- v# G
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Fig. 2. Distribution of COX-1 IR in cryostat sections of rat and mouse renal cortex as revealed by double-labeling or consecutive-sectioning techniques using segmental markers of the nephron combined with different fluorochromes: Tamm Horsfall protein (THP) for thick ascending limb (TAL; ); thiazide-sensitive Na-Cl cotransporter (NCC) for the distal convoluted tubule (DCT;°); and Na/Ca exchanger (Na/Ca) for the distal convoluted tubule (DCT) and the connecting tubule (CNT; * ). Insertions of white bars indicate the junctions between segments and subsegments of the distal convolutions according to previous mapping ( 3, 6 ). a, b, g, and h : COX-1 is absent from THP-positive TAL. c, d, i, and j : COX-1 is absent from early DCT but present in late DCT and CNT. e, f, k, and l: COX-1 is present in terminal DCT and CNT. CD, collecting duct. Approximate magnification: x 400.
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Fig. 3. Distribution of COX-1 in CNT ( * ) and CD ( ) in rat, w.t. mouse, and COX-1 k.o. mouse renal cortex using immunoperoxidase staining. a, c, and e : single CNT profiles are seen in the vicinity of cortical arterioles. d : ultrastructural labeling of a CNT cell with enhanced perinuclear signal; note an adjacent, negative intercalated cell (#). g and h : In the k.o. mouse, absence of staining is seen. Paraffin sections, hematoxylin stain, and interference contrast ( a, b, and e - h ); semithin section ( c ); and ultrathin section ( d ) are shown. Approximate magnifications: x 400 ( a ); x 800 ( b, e - h ); x 1,000 ( c ); and x 5,500 ( d ).
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9 e+ J! ^9 |0 C3 s# M* SFig. 4. Distribution of COX-1 and PGE synthase (PGES) in mouse cortical interstitial cells (arrowheads). Paraffin sections with hematoxylin counterstain ( a ) and interference contrast ( b ) are shown. Approximate magnification: x 1,000.) v1 C0 X0 K0 T8 J( G/ C
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Fig. 5. In situ hybridization of paraffin sections showing COX-1 mRNA in rat and mouse kidney. Segmental markers of the nephron (NCC mRNA for DCT;°) and IR calbindin for DCT and CNT ( * ) are shown in consecutive sections. COX-1 mRNA partially colocalizes with NCC mRNA in rat kidney ( a and b ) and with calbindin in mouse kidney ( c and d ). Approximate magnifications: x 400 ( a and b ) and x 800 ( c and d )., n) _) ?3 L8 W! f! j" r
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Compared with COX-1 immunolocalization, COX-2 expression in rat renal cortex was not found in the distal convolutions, but, rather, it was restricted to scattered single cTAL cells or smaller groups of cells, thus confirming earlier work ( 17, 45 ) ( Fig. 6, a and b ). A COX-2-immunoreactive signal was concentrated in the macula densa region of cTAL, although macula densa cells proper were rarely stained ( Fig. 6, a and f ). In situ hybridization showed similar distribution of positive cTAL cells ( Fig. 6 c ). Ultrastructurally, COX-2 immunostaining of macula densa cells proper revealed prominent staining of the nuclear envelope and little cytosolic labeling, whereas in adjacent or upstream locations of cTAL, positive cells showed predominantly cytosolic labeling that extended into the interdigitating cell processes ( Fig. 6, d and e ). In mice, COX-2 staining was different from that in rats, with a signal mostly confined to the macula densa ( Fig. 6 h ). Apart from TAL, all other structures of the nephron, glomerulus, and interstitium were negative. Generally, vascular COX-2 immunostaining was not observed. The COX-2 knockout mouse did not show renal COX-2 immunoreactivity either (data not shown).5 `( Y8 q$ H6 X1 V1 ], x* ^
# M, m) V/ [. M( e% {/ }+ HFig. 6. Distribution of COX-2 in rat and mouse renal cortex. a and b : Juxtaglomerular ( a ) and further-upstream portions ( b ) of cortical TAL (cTAL) and macula densa (between arrowheads) showing COX-2 immunofluorescence [red or yellow; double-stained with anti-THP antibody (green) to mark TAL]. c: In situ hybridization reveals staining in analogous location. d and e : Ultrastructural immunoperoxidase labeling shows prominent nuclear envelope staining in macula densa cells ( d ) and predominant cytosolic staining extending in cTAL cell processes ( e ). f and g : PGES immunoperoxidase staining is restricted to macula densa and adjacent cTAL cells ( g ), whereas concomitant COX-2 staining in macula densa may be weak as shown ( f ). h and i : COX-2 in mouse macula densa colocalizes with PGES. G, glomerulus. Approximate magnifications: x 400 ( a, f, and g ); x 600 ( b, h, and i ); x 300 ( c ); x 3,600 ( d ); and x 2,200 ( e ).
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We next investigated the distribution of PGES in rats and mice to establish whether this downstream-acting enzyme would be associated with one or both of the COX isoforms. PGES staining in glomeruli was restricted to occasional single cells that were not identified. In agreement with COX-2 localization, PGES immunoreactivity was detected in rat terminal cTAL portions, macula densa, and the short postmacular segment in rats ( Fig. 6 g ), and in mice, a signal was restricted to the macula densa, in agreement with COX-2 localization ( Fig. 6 i ). The DCT1 was entirely negative in rats, whereas PGES staining in the terminal DCT, CNT, and CCD was intensive; in mice, an analogous distribution was encountered ( Fig. 7, a - h ). Double staining for PGES and COX-1 or COX-2 confirmed these results (Figs. 6, f and g, and 8, a - h ). In mice, a major proportion of the cortical interstitium showed PGES-immunoreactive interstitial fibroblasts in the cortical labyrinth, which is in agreement with COX-1 localization ( Fig. 4 b ); in rats, this pattern was far less conspicous.5 @: a0 ?* n( Q2 v7 t+ N
% ?- s: c2 Y, d* M6 [Fig. 7. Distribution of microsomal PGES in rat and mouse renal cortex. Consecutive sections are stained for PGES and for NCC as a marker of DCT (°) and calbindin as a marker of DCT and CNT ( * )., Collecting duct. Immunoperoxidase staining in rat, immunofluorescence staining in mouse. Approximate magnification: x 200.
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Fig. 8. Comparative distribution of COX-1 and PGES in rat kidney cortex and medulla immunoperoxidase stained on consecutive sections. There is complete overlap of signals in CNT ( * ) and in medullary collecting duct profiles of different levels ( ). Note COX-1 and PGES signals also in the papillary surface epithelium (arrowheads in g and h ). Approximate magnification: x 400.
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Medulla. Significant epithelial COX-1 immunostaining was detected in the principal cells of collecting ducts from outer and inner medulla in rats and mice ( Fig. 9, a and d ). Medullary collecting duct (MCD) staining extended all the way from the outer to inner medulla and papilla and was also found along the papillary surface, but not in the pelvic epithelium; this pattern was also found for PGES immunoreactivity ( Fig. 9, c and f ), except for the pelvic epithelium, which was strongly PGES positive. COX-1 in situ hybridization produced only a faint signal in MCD portions compared with CNT or CCD, indicating lower mRNA levels at these sites (data not shown). COX-1 immunostaining was also observed in the majority of the outer medullary interstitial cells in mice, whereas in rats interstitial cells of this portion were rarely stained. In the papillary portion of the inner medulla, however, both species showed strong perinuclear COX-1 staining of the lipid-laden interstitial cells, except for a small terminal portion of the papillary tip interstitium./ S# E7 W3 W& G+ V+ W
4 v3 t: k- w$ eFig. 9. Comparative distribution of COX-1, COX-2, and PGES in rat and mouse renal inner medulla. MCD ( ) and interstitial cells may be distinguished in all sections. Note the absence of COX-2 from MCD profiles ( b and e ). Paraffin sections partly counterstained with hematoxylin are shown. Approximate magnifications: x 1,000 ( a and e ), x 600 ( b, c, and f ), and x 400 ( d ).7 ]' s+ C3 C* }
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By contrast, medullary COX-2 staining produced no signal in the collecting ducts in either species ( Fig. 9, b and e ). The lipid-laden inner medullary interstitial cells of the inner medulla and papilla, however, showed significant COX-2 immunoreactivity, although interindividual variability was high. In the papillary tip in rats, interstitial cells were mostly negative. In mice, COX-2 staining along the papillary interstitium was scarce. However, a remarkable increase in staining was incidentally observed when mice had been exposed to surgical stress (laparotomy and exposition of kidney during several hours).
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5 j. h9 F$ s& ^. O1 cPGES-immunoreactive cells in the rat and mouse medulla were distributed analogously to COX-1-expressing cells. Significant epithelial PGES staining was detected in the principal cells of MCD from outer and inner medulla ( Fig. 9, c and f ). The distribution of COX isoforms and PGES is summarized ( Fig. 10 and Table 1 ).2 o* |& n% T3 q% a2 M! q
5 ?+ Z9 L9 `& J+ R( s( N; [5 H. Y* _5 rFig. 10. Schematic topographical representation of COX-1 ( A )-, COX-2 ( B )-, and PGES ( C )-immunoreactive sites (shaded areas) in rat kidney. With the exception of the COX-2 signal in the TAL/macula densa region, this distribution is also found in mouse kidney.5 ?! N+ W: l7 s/ ]9 v. Y6 e
$ o9 H: w- X2 D( H3 Q( D+ Y; P" n4 _
Table 1. Distribution of COX isoforms and PGES in rat and mouse kidneys
; A& p: w1 h P: H) d5 s3 I% {+ T0 i; ~, A- q( h3 T( {
Complementary localization of COX isoforms in knockout mice. Localizing COX-1 or COX-2 expression in the respective complementary knockouts showed no principal differences in the typical position or signal intensity of the respective mRNA or immunoreactive staining ( Fig. 11 ).: `7 E$ k# X. u1 K( b9 E1 I. l- M
% c4 x+ l( N5 a* a- D4 q& AFig. 11. Representative images of a comparative localization of COX-1 expression in a COX-2 knockout mouse ( a and c ) and of COX-2 expression in a COX-1 knockout mouse ( b and d ). a and b : in situ hybridization with radiolabeled probes showing darkfield overview of kidneys with typical COX-1 mRNA expression in CNT, CCD, and MCD and medullary interstitial cells. c and d : immunofluorescence staining showing typical distribution of COX-1 in connecting tubules and COX-2 in macula densa cells. Approximate magnifications: x 50 ( a and b ) and x 400 ( c and d ).
3 k# [) T$ k& U5 ~" a& O4 {* s& R& `( o# m* s6 Y8 V
RT-PCR analysis. The expression of COX-1, COX-2, and PGES mRNA was detected in extracts from rat glomeruli. GAPDH was used as a positive control ( Fig. 12 A ).
/ M6 S; K& h) t' Y) W7 z3 ?
* k* c; i5 E0 N' D9 o1 B1 O- zFig. 12. A : representative RT-PCR analysis of COX-1, COX-2, and PGES mRNA in extracts from isolated rat glomeruli with GAPDH as a reference standard. B - D : representative Western blots for COX-1, COX-2, and PGES, respectively, from protein extracts of whole rat and mouse kidneys.9 I/ G7 \6 X3 ?" C8 p, I" H1 g
0 V2 @9 T: \2 f. M' V7 E3 f5 hWestern blot analyses. Western blotting was performed to confirm antibody specificity. COX-1 immunoreactivity showed a single, distinct band at 70 kDa, COX-2 at 72 kDa, and PGES at 17 kDa ( Fig. 12, B - D ).
v5 F; ?6 f7 M! N
( W0 l9 [, Z/ I* V8 R" e( cDISCUSSION; [2 m& f' k0 n: H( W1 X- i
! ]) E% N$ z5 y) L* f
In this study, we have presented a comprehensive analysis of the expression sites of enzymes involved in renal prostaglandin synthesis using histochemical techniques for cell-type identification. Among several commercially available antisera labeled anti-COX-1 or anti-COX-2 raised against peptides, we have made a careful choice after having applied rigorous criteria for specificity, and we have corroborated our results by in situ hybridization and the use of knockout mice.1 N4 \" a& o& [! L) s
9 Z) a$ l1 w; @8 j! i& ]Glomerulus. In the renal corpuscle, COX-1 was the dominant isoform in rats to be expressed in a subset of glomerular mesangial cells. This agrees with previous results by Soler et al. ( 44 ) reporting the release of untransformed PGH 2 as the major product from cultured mesangial cells. Because we could not detect the downstream-acting PGES in mesangial cells, it appears plausible that this precursor prostanoid is released also in vivo from the glomerular tuft. With respect to nephritic conditions, stimulated COX-1 levels together with increased EP2 receptor expression have been suggested to be potentially beneficial ( 21 ). In the human kidney, immunoreactive COX-1 has been localized to podocytes during development, and to endothelial cells of the glomerular hilus ( 31 ); these cell types, however, were not positive in our studies. In contrast to COX-1, immunoreactive COX-2 was not detected reliably in the glomerulus, although COX-2 mRNA was clearly detectable using RT-PCR. The absence of COX-2 protein in glomerular extracts from control tissue was also reported in another study by Western blot analysis ( 10 ), and even under inflammatory conditions no immunoreactive COX-2 was detected in podocytes ( 30 ). The rodent glomerulus therefore appears to be a minor site of prostanoid synthesis originating from the action of mesangial COX-1, but in turn it may well be a sensitive target for local prostaglandins because podocytes express various prostanoid receptors ( 4 ).
8 \; N5 P5 Q' s" w0 |$ v \# E5 M+ h' g: y9 ]
Loop of Henle. Along the nephron, the first evidence for the parameters tested was found in the loop of Henle of rat kidney with COX-2- and PGES-expressing portions of cTAL and macula densa. By contrast, COX-1 was absent from these cells, confirming earlier work ( 17, 42 ). COX-2 expression in rats differed from that in mice because a COX-2 signal in rats was typically found in scattered individual cells or cell groups along the cTAL and in a small proportion of macula densa, as established elsewhere ( 23, 45, 46, 51; for a review, see also Refs. 17, 19, and 40 ), whereas in mice, COX-2-positive cells were less numerous and concentrated in macula densa and a few directly adjacent TAL cells. The total number of COX-2-positive TAL cells in normal rats has been shown elsewhere to amount to " C x& h' u2 o4 ~8 Z% \
a$ U4 v# o$ O {, q* |! J D
Numerous studies on experimentally induced changes of COX-2 activity have used biochemical assays on tissue homogenates for quantitative evaluation. However, regarding the paucity of cellular expression and the heterogenous distribution of COX-2-positive cells, the present study underlines that histochemical quantification establishing the number and size of COX-2 loci, or the number of positive individual cells related to volume of tissue, should result in a more reliable evaluation. Micromorphological distinctions between macula densa and cTAL COX-2 expression may thus be respected ( 17, 45, 51 )., ?- ?2 t! H( ]+ v2 h5 b
% F2 p s* o% I" fThe action of COX-2 in cTAL is relevant to essential functions of the epithelium itself and of the tubulovascular signaling mechanism that reacts to the local release of PGE 2, the major prostaglandin from TAL ( 40 ). On one hand, COX-2-derived prostanoids seem to be permissive for the effect of loop diuretics ( 27 ) and exert an inhibitory effect on Na-K-Cl cotransport in mTAL cells ( 26 ). It has accordingly been established that PGE 2 directly inhibits solute reabsorption in in vitro perfused mTAL, although data on cTAL are less clear cut, and the identification of an E-type prostaglandin receptor in cTAL is still controversial ( 5, 25 ). On the other hand, there is firm evidence that low chloride activates COX-2 in TAL and macula densa (for a review, see Ref. 40 ); in cultured macula densa cells, a correlation between activated COX-2 and increased prostaglandin release has been established ( 48 ). The latter seems to be a critical component in macula densa control of renin synthesis and of its release. This was particularly evident from hyperreninemic states resulting from furosemide treatment, renal arterial stenosis, or loss-of-function mutations of Na-K-Cl transport components in TAL, which were paralleled by a recruitment of COX-2-positive cells ( 40 ). An interaction with the renin-angiotensin system is also well recognized, because an ANG II-dependent negative-feedback effect on tubular sites of COX-2 synthesis has been reported, and mice that are nullizygote for ANG II receptor subtypes expressed high levels of COX-2 in macula densa ( 8 ). Our finding of intensive, constitutive COX-1 expression in the extraglomerular mesangial cells in this context is intriguing, although its particular relevance in the regulatory physiology of the juxtaglomerular apparatus remains to be established. Local release of prostaglandins from both sources may thus affect glomerular microcirculation and control of glomerular filtration rate, although controversy as to the particular receptors involved must still be resolved.9 w" T* a0 ~: y
& a6 c% F. B6 k, y, WIt has been established that the expression of COX-2 during development is much stronger than in the adult rodent kidney. This has also been evaluated quantitatively ( 51 ). Regarding our observation that in mouse cTAL, the COX-2 signal is proportionately less extended than in rat cTAL, it is noteworthy that COX-2 gene disruption in mice nevertheless causes major damage to nephron development ( 33 ); this underlines the prominent role of perinatal prostaglandin synthesis at the juxtaglomerular site at least in the rodent kidney, because no other COX-2-immunoreactive cell type was detected in cortex.# l' x% e; i. j2 g7 @
" Q2 o- `/ w0 k7 G5 T) |- c6 C7 uFine structural analysis showed the typical perinuclear location of immunoreactive COX-2, which was also found for COX-1 in mesangial cells, thus confirming that both COX isoforms predominantly integrate into the outer leaflet of the nuclear membrane; however, additional cytoplasmic label was observed as well in cTAL cells, including their fine cellular processes, suggesting that COX-2 may also exert its effects in membrane compartments distant from the nucleus. This finding proves in more detail what others have stated earlier in the neonatal ( 51 ) and adult ( 23 ) rat kidney using distinct methods. The intracellular signaling events related to the action of COX-2 in cTAL have partly been identified. Rapid phosphorylation of p38 and ERK1/2 kinases and stimulation of MAP kinase pathways are involved ( 7, 48 ). Expression of microsomal-type PGES in cTAL and macula densa suggests that the formation and release of PGE 2 at these sites are functionally coupled to the action of this enzyme.( O z9 P; k& H4 `. O4 U+ S1 G) m4 v
5 h* X. M! y! @- J( h2 E1 j( _4 [( c
Distal convolutions and collecting duct. Expression of COX-1 in the collecting duct has been described previously ( 42, 47 ), but evidence for detailed protein localization in the distal convolutions was missing. We have shown that the portion immediately after the postmacula segment of the TAL, i.e., the initial DCT, was devoid of COX, whereas in the terminal DCT portion, significant COX-1 staining was obvious in both species, and expression continued into the CNT, CCD, and MCD portions, and was accompanied throughout by significant PGES immunoreactivity in both species. By contrast, intercalated cells in our hands were never stained for any of the products investigated in either species, which is at variance with observations by Ferguson et al. ( 11 ) reporting COX-1 and COX-2 signals only in intercalated cells of the CCD. Probe-specificify problems may be the reason for this discrepancy., w, V) u3 ]( A" f2 E& o# S
, n/ _! R/ i7 p$ L. _5 E0 \Endogenously formed or exogenously applied PGE 2 is well known to reverse the effect of antidiuretic hormone (ADH) in the collecting duct. When added to vasopressin-prestimulated collecting ducts, it potently inhibits water absorption ( 22 ), and it may also reduce sodium reabsorption in the collecting duct via a calcium-coupled mechanism ( 14 ). By contrast, basolateral effects of PGE 2 may also include a stimulation of water reabsorption in this segment ( 22 ). There is further evidence that in inner MCD Na-K-ATPase activity was diminished by PGE 2. Receptors in the collecting duct that mediate these local effects are essentially EP1 and, with spacial restrictions, EP3, as shown pharmacologically and by expression analysis ( 5, 25 ). It is not clear why the late DCT and the CNT show the strongest signal of all renal epithelia for COX-1 and PGES. Local effects may be mediated via the EP4 receptor ( 25 ) and may interfere with local hormonal systems such as the kallikrein-bradykinin system ( 36 ) or with the function of aldosterone, because these segments are sensitive to mineralocorticoids and express the amiloride-sensitive epithelial sodium channel (for a review, see Ref. 3 ). As in the collecting duct, COX-1 action in CNT may interfere with the effects of ADH. The pronounced coexpression of COX-1 and PGES in the pelvic epithelium underlines the similarity to the MCD, although the functional significance of this observation is not clear. Previous work has demonstrated the distribution of a mouse PGES form in distal tubule using in situ hybridization and immunostaining; labeling was concentrated in Tamm-Horsfall-negative distal tubules, which is compatible with the present findings ( 15 ).4 G% x! j* p$ @
% l( H! u/ s9 G" l0 W$ d" H; YInterstitial cells. The renal interstitium has long been recognized as the major source for prostaglandin biosynthesis, with the medulla as the principal location. However, in mice we also found strong immunoreactivity for COX-1 in cortical interstitium. Rats failed to show a clear signal at this site, although interstitial PGES was found in both species, suggesting PGE 2 release by these cells. Medullary interstitial cells were shown to abundantly and constitutively express both COX isoforms in rat, whereas COX-2 in mice was infrequent. Our findings are in agreement with previous work in rats and humans ( 31, 37, 51 ) and extend findings to mice. Prostaglandin synthesis in the medulla adjusts local microcirculation (reviewed in Ref. 5 ). Relevant effectors in this context may be the EP2 or EP4 receptors located in the vasa recta to mediate the response to high-salt intake and protect against systemic hypertension ( 28 ). The overlap of COX-1 and -2 expression in rats is puzzling and makes a distinction between in vivo contributions of the isoforms difficult; this is further hampered by the segregated COX-1 expression in the MCD; these questions have partly been addressed recently with respect to hormonal influences and intracellular localization specificities in rats ( 50 ). The coexpression of PGES with COX in the interstitial cells further supports in vitro data that the major prostanoid resulting from local COX activity may indeed be PGE 2 ( 16 ). The particularly well-identified role of the action of COX-2 comprises its response to physiological renal stress such as water deprivation, and it has further been shown that COX-2 allows the interstitial cells to survive hypertonic stress after dehydration ( 49 ).
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Other structures. In contrast to other reports, vascular labeling with antibodies to COX isoforms was not reliably detected, except for an occasional endothelial COX-1 signal. In general, regarding COX-2 expression, the reader should be aware that we have reported only the constitutive localization of COX-2, whereas under inflammatory conditions or induction by altered steroid levels, many more cells and cell types may express COX-2 in the kidney as elsewhere in the body ( 46; for a review, see also Ref. 13 )./ ~/ [+ w0 }$ c+ X) }% ~: o
2 b7 i9 ~3 U, i" R) `0 }Complementary localization of COX isoforms in knockout mice. The lack of obvious changes in the localization of COX-1 and COX-2 expression in the respective, complementary knockouts suggests that deficiency of one isoform has a major compensatory impact on the expression of the other.' T( Z, Z' h+ |- l- E5 l
: ^( Q l; W' c& D4 ~
In sum, our data underscore prominent functions of renal prostaglandin synthesis by defining the expression sites of the key enzymes involved in their biosynthesis. For this purpose, data raised in rats were compared with those in mice to broaden the knowledge for experimental settings that make use of knockout mice. Results define the renal cell types involved in presumed prostaglandin release within spatially restricted sites such as the juxtaglomerular apparatus, mesangium, distal convolutions and collecting duct, and in compartments of the renal interstitium.
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. P! T! e$ K* n/ h5 k$ xACKNOWLEDGMENTS
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We thank Hermann Pavenstädt (Dept. of Nephrology and Internal Medicine, University of Freiburg, Freiburg, Germany) for providing us with rat glomerular mRNA extracts. Technical help in immunohistochemistry by Kerstin Riskowsky and Gudrun Holland is gratefully acknowledged.4 k8 ^3 l1 ?; H6 I: F; A
% b! i; W8 s0 t5 }9 x# VThis work was supported by funds from the Deutsche Forschungsgemeinschaft (Ba 700/14-1 and 14-2).
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发表于 2009-4-21 13:51
