Cyclooxygenase-2 in rat nephron development

Ming-Zhi Zhang1, Jun-Ling Wang2, H.-F. Cheng2, Raymond C. Harris2, and James A. McKanna1

George M. O'Brien Kidney and Urologic Diseases Center and 1 Department of Cell Biology, 2 Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

The inducible second isoform of cyclooxygenase (COX-2) that mediates inflammation also is expressed at low levels in normal adult rat kidneys and is upregulated in response to noninflammatory stimuli (R. C. Harris, J. A. McKanna, Y. Akai, H. R. Jacobson, R. N. DuBois, and M. D. Breyer. J. Clin. Invest. 94: 2504-2510, 1994). Roles in morphogenesis are indicated by reported teratogenicity of COX inhibitors and renal dysgenesis in COX-2 knockout mice (J. E. Dinchuk, B. D. Car, R. J. Focht, J. J. Johnston, B. D. Jaffee, M. B. Covington, N. R. Contel, V. M. Eng, R. J. Collins, P. M. Czerniak, A. G. Stewart, and J. M. Trzaskos. Nature 378: 406-409, 1995; S. G. Morham, R. Lagenbach, C. D. Loftin, H. F. Tiano, N. Vouloumanos, J. C. Jennette, J. F. Mahler, K. D. Kluckman, A. Ledford, C. A. Lee, and O. Smithies. Cell 83: 473-482, 1995). Blots from developing rat kidneys demonstrated that COX-2 mRNA and immunoreactive protein were present in neonates, peaked in the 2nd and 3rd postnatal weeks and declined to adult levels by the 3rd month. Immunolocalization and in situ hybridization detected intense COX-2 immunoreactivity and mRNA in a subset of thick ascending limb epithelial cells near the macula densa in each developing nephron; after 2 wk the COX-2 gradually waned. These data demonstrate that COX-2 expression is subject to normal developmental regulation and can be sustained over extended periods; they also support the conclusion that metabolites of COX-2 play important roles in the differentiation and early functions of mammalian nephrons.

kidney; nephrogenesis

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

IN ADULT MAMMALIAN KIDNEY, prostaglandins regulate renal hemodynamics and salt and water homeostasis. Prostaglandin production depends on the release of arachidonic acid from membrane phospholipids by specific phospholipases and subsequent conversion to prostaglandin H2 (PGH2) by PGG2/H2 synthase [also known as cyclooxygenase (COX)]. Further metabolism by specific synthases produces individual prostanoid species (25). Two distinct COX genes have been described: a "constitutive" form (COX-1) that encodes a 2.7- to 2.9-kb transcript (4) and an "inducible" form (COX-2) that encodes a 4.0- to 4.5-kb transcript (6, 12).

In the kidney, COX-1 immunoreactivity (COX-1-ir) has been localized to arteries and arterioles, glomeruli, and collecting ducts (33); no COX-1-ir has been found in the proximal or distal convoluted tubules, Henle's loop, or macula densa. In adult animals, COX-2 expression has been seen predominantly during cell growth or in inflammatory states (27, 16, 20). Accordingly, COX-2 expression would not be predicted in normal kidney.

However, we recently reported that low but measurable levels of COX-2 mRNA and protein were detectable in normal adult rat kidney (9). In situ hybridization and immunohistochemistry demonstrated strong COX-2 expression in sparse scattered cells of the cortical thick ascending limb (CTAL) of Henle's loop near the macula densa. Furthermore, after depletion of dietary sodium chloride, the numbers of cells expressing COX-2 in the TAL increased significantly. These results indicated that COX-2 expression can be regulated by noninflammatory factors and that expression of COX-2-ir in this region of the nephron is not transitory.

In addition, recent studies utilizing targeted disruption of COX-1 or COX-2 gene expression have indicated that COX-2 is important for normal metanephric development. No alterations in renal structure or function were detected following disruption of COX-1 gene expression (13); however, significant abnormalities of kidney development appeared in mice with targeted disruption of COX-2 expression (5, 22). Therefore, the present studies were designed to elucidate temporal, spatial, and cytological aspects of COX-2 expression during fetal and postnatal kidney development.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Sprague-Dawley male and Long-Evans female rats were used to produce timed hybrid pregnancies. Noon of the sperm-positive morning was designated as embryonic day 0.5 (E0.5); the day of birth was designated postnatal day 0 (P0).

Immunoblotting. Kidneys were homogenized in 30 mM tris(hydroxymethyl)aminomethane (Tris) hydrochloride, pH 8.0, 100 µM phenylmethylsulfonyl fluoride (1:9 wt/vol). Following a 10-min centrifugation at 10,000 g, the supernatant was centrifuged for 60 min at 110,000 g to prepare microsomes as described previously (9). The microsomes were resuspended in sodium dodecyl sulfate (SDS) sample buffer and heated to 100°C for 5 min, and the proteins were separated on 8% SDS gels under reducing conditions and transferred to Immobilon-P transfer membranes (Millipore, Bedford, MA). The blots were blocked overnight with 100 mM NaCl-50 mM Tris-Cl, pH 7.4, containing 5% nonfat dry milk, 3% albumin, and 0.5% Tween-20, followed by incubation for 16 h with rabbit polyclonal antiserum raised against a murine COX-2 peptide (Cayman Chemical, Ann Arbor, MI) at 2.5 µg/ml dilution. The second reagent, biotinylated goat anti-rabbit antibody, was detected using avidin and biotinylated horseradish peroxidase (Pierce, Rockford, IL) and exposed on film using enhanced chemiluminescence (ECL, Amersham).

Northern analysis. Total kidney RNA was isolated by the acid guanidinium thiocyanate-phenol-chloroform method (2). RNA samples were electrophoresed in denaturing agarose gels and transferred to nitrocellulose. Nitrocellulose blots were hybridized as previously described with a 1.3-kb 32P-labeled cDNA Kpn 1/Xho 1 fragment of the 3'-untranslated region (UTR) of rat COX-2 (9).

Immunohistochemistry. Under deep anesthesia with Nembutal (70 mg/kg ip), rats were exsanguinated with ~50 ml/100 g heparinized saline (0.9% NaCl, 2 U/ml heparin, 0.02% sodium nitrite) through a transcardial aortic cannula and fixed with glutaraldehyde-periodate-acid-saline (GPAS) as previously described (19). GPAS contains final concentrations of 2.5% glutaraldehyde, 0.01 M sodium m-periodate, 0.04 M sodium phosphate, 1% acetic acid, and 0.1 M NaCl; it provides excellent preservation of tissue structure, COX-2 antigenicity, and mRNA. The fixed kidneys were dehydrated through a graded series of ethanols, embedded in paraffin, sectioned at 4 µm thickness, and mounted on glass slides. COX-2-ir was immunolocalized with polyclonal rabbit anti-murine COX-2 serum (Cayman) diluted to 2.5 µg/ml. The primary antibodies were localized using Vectastain ABC-Elite (Vector, Burlingame, CA) with diaminobenzidine (DAB) as chromogen, followed by a light counterstain with toluidine blue.

In situ hybridization. Beginning with the saline exsanguination, all solutions for this protocol were prepared with deionized autoclaved water containing 0.1% diethyl pyrocarbonate to destroy ribonucleases (RNases). GPAS as described above was as good or better than all other fixatives examined. Prior to hybridization, sections were deparaffinized, treated with proteinase K (5 µg/ml) for 20 min, washed with phosphate-buffered saline, refixed in 4% paraformaldehyde, and treated with 0.1 M triethanolamine (pH 8.0) plus acetic anhydride (0.25% vol/vol), and then dehydrated through a graded series of ethanols.

The sense and antisense probes were synthesized by linearizing the 1.3-kb 3'-UTR rat COX-2 fragment ligated into pBSK(-) and transcribing from the flanking T7 or T3 promoters in the presence of digoxigenin-UTP. The probes were hybridized to sections at 55°C for 18 h, as previously described (9). Sections were washed at 50°C in 5× SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) for 10 min, followed by washes in 50% formamide + 2× SSC twice for 30 min, then in 10 mM Tris + 5 mM EDTA + 500 mM NaCl (TEN) twice for 10 min. Sections were then treated with RNase (5 µg/ml) at 37°C for 30 min, followed by TEN, twice at 50°C with 2× SSC, and once with 0.2× SSC. Hybridization was detected using a monoclonal anti-digoxigenin immunoglobulin G-alkaline phosphatase complex (Boehringer Mannheim). No hybridization by sense RNA was detected.

Quantitative image analysis. Based on the distinctive density and color of COX-2-ir in video images, the number, size, and position of stained cells were quantified using the BIOQUANT true-color windows system (R & M Biometrics, Nashville, TN) equipped with digital stage encoders that allow high-magnification images to be mapped to global coordinates throughout the whole kidney. Sections from at least three regions of each kidney were analyzed. Foci of antisense mRNA probe hybridization also were analyzed using this system.

Micrography. Bright-field images from the Leitz Orthoplan microscope with Optronics DEI750 three-chip red-green-blue color video camera were digitized by the BIOQUANT TCW system and saved as computer files. Contrast and color level adjustments (Adobe Photoshop) were performed for the entire image; i.e., no region- or object-specific editing or enhancements were performed.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Northern analysis. Consistent with our previous observations (9), a cDNA probe specific for rat COX-2 detected low-level expression of the 4.4-kb message in control kidneys from a normal adult male rat ~10 mo of age (Fig. 1). As calibrated per gram of tissue by comparison with the constitutive glyceraldehyde-3-phosphate dehydrogenase message, developmental regulation of renal COX-2 expression was apparent in rat pups. The signal in newborns (P0) was similar to adult; but by P1 the signal was stronger, and by P3 COX-2 mRNA was substantial. The signal remained strong from P7 through P14 but began to decrease by P21 (not illustrated)


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Fig. 1.   Northern analysis. Total mRNA from kidneys of rats staged from newborn [postnatal day 0 (P0)] to adult (A). Probe for cyclooxygenase-2 (COX-2) mRNA hybridizing to distinct band at 4.3 kb (top) demonstrates upregulation by day P1 and strong expression by P3 and P7. Signal for the constitutive protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), confirms equivalent loading for all lanes.

Western analysis. Expression of COX-2 protein in developing and adult rat kidneys was examined with immunoblots using serum raised against a peptide from murine COX-2. The immunoreactive COX-2 in the microsome fraction from whole kidney homogenates appeared as a distinctive band of ~73 kDa (Fig. 2). In lanes loaded with equal protein, the density of the COX-2 band was low at P0, increased gradually to reach a peak at P14, and decreased gradually to very low levels in the adult.


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Fig. 2.   Western analysis. Microsome fraction from total kidney homogenates of rats staged from newborn (P0) to adult (A). Equivalent amounts of protein loaded in each lane were separated by gel electrophoresis and transferred to a membrane for immunochemical identification with anti-COX-2 serum. COX-2 immunoreactivity (COX-2-ir) peaked at P14 and declined to low levels in adult.

Immunohistochemistry. In the developing metanephric kidneys from embryonic day 16 (E16) rats, diffuse cytoplasmic COX-2-ir was apparent in cells of both the branching collecting ducts derived from ureteric buds and the S-shaped bodies induced from mesenchyme (Fig. 3A). The staining filled the cytoplasm of these cells, showing no preference for either the supra- or infranuclear domains (Fig. 3B). Although the COX-2-ir at this stage was diffuse, the brown DAB reaction product contrasted clearly with the dull blue COX-2-negative staining of the uninduced mesenchyme and embryonic connective tissue cells. This diffuse mode of expression was observed in successive layers of the outer cortex as nephrogenesis progressed through the second postnatal week. From early stages, COX-2-ir was restricted predominantly to the metanephros; it was not evident in mesonephros or other embryonic structures (not illustrated).


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Fig. 3.   A and B: embryonic day 16 (E16) epithelial cells of both ureteric origin [collecting ducts (d), branching buds (b)] and mesenchyme [S-shaped body (s), nephron (n), glomerulus (g)] display the light brown staining of diffuse COX-2-ir. Subcapsular uninduced mesenchyme and interstitial connective tissue cells are COX-2 negative. Bar in A is 125 µm. C-F: at embryonic day 20 (E20), intense COX-2-ir is restricted to tiny foci associated with the juxtamedullary nephrons that are known to be functional at this time (C, arrowheads). At higher magnifications, it is apparent that the COX-2 foci are at the vascular poles of glomeruli (g) and are individual cells (D, arrows) that are part of the epithelium of the thick ascending limb (tal). Higher magnifications show that the COX-2-cells are adjacent to but not part of the macula densa (m). Although it is not clear that the diffuse stained COX-2 cells give rise to the intense ones, intermediate forms can be observed (E, arrow). Relative sizes of scale bar in A are 125 µm (A), 40 µm (B), 500 µm (C), 125 µm (D), 40 µm (E), and 30 µm (F).

By late gestation (E20), a cytologically distinctive intense mode of COX-2 expression was observed (Fig. 3, C-F). The immunohistochemical reaction product in these COX-2 cells filled the cytoplasm and sometimes obscured the nucleus, but in most planes of section it was apparent that the nucleus was not stained. This mode resembled the COX-2-ir pattern described previously for adult rat kidneys (9).

The first generation of intense COX-2 cells was observed in the early juxtamedullary nephrons (Figs. 3, C and D). At this time, the TAL epithelium displayed diffuse COX-2-ir as described above, but a few cells near the nascent macula densa exhibited much stronger COX-2 staining. Intermediate graded stages between the diffuse and intense COX-2-ir were not abundant but were seen occasionally (Fig. 3E, arrow).

Examination of various planes of section indicated that the cells with intense COX-2-ir were perimacular; i.e., they were proximal to and distal to and across from the macula densa, but the macula cells per se were COX-2 negative (Figs. 3F and 4B). Intense COX-2-ir apparently filled the entire cytoplasm of individual cells in the epithelium of the TAL (Fig. 3, E and F). Although COX-2 cells in the epithelium often were contiguous, many individual/isolated COX-2 cells also were observed as the thick limb matured. At the interfaces with contiguous COX-2-negative cells, the basal interdigitations of COX-2 cells were revealed in stark contrast (Fig. 4D), indicating that the pervasive COX-2-ir extended into the delicate mitochondria-rich basal folds that interdigitate with adjacent cells. On the basis of these morphological criteria, the COX-2 cells clearly are characterized as bona fide members of the tubular epithelium rather than transient migrant cells of a different lineage, e.g., monocytes or lymphocytes.


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Fig. 4.   Left: comparison of COX-2-ir in the three low-magnification images of cortex at successive stages of kidney morphogenesis reveals initial juxtamedullary loci near the arcuate arteries (a) at day P7 (A), progressing to the medullary rays and subcapsular region at P15 (C), and diminishing toward adult levels by P39 (E). Right: as a juxtamedullary glomerulus matures (B), cells of the thick ascending limb (TAL) at the vascular pole show intense COX-2-ir all around the lumen except for the differentiating specific cells of the macula densa (arrows); by P15 (D), individual maturing cells of the cortical TAL in the medullary rays contain intense COX-2-ir that stains their basal interdigitations (arrows); higher magnification (F) of boxed region in E shows that by P39, the COX-2 cell population has diminished to comprise only isolated cells in subcapsular thick limbs (arrows). Relative sizes of Fig. 3A scale bar: 500 µm (A, C, E), 30 µm (B, D), and 125 µm (F).

Normal kidney morphogenesis progresses centrifugally through subcapsular induction of successive generations of nephrons. Accordingly, as new nephrons differentiated, the pattern of COX-2 expression originally apparent in the juxtamedullary region gradually moved further out in the cortex (Fig. 4, A and C). The cell population displaying intense COX-2-ir waxed and waned over a period of 4-5 wk as each nephron developed. Shortly after the glomerulus was vascularized, the first COX-2 cells appeared at the future site of the macula densa. Subsequently, COX-2 cell numbers in the growing TAL continued to increase for 7-10 days. Because of the considerable increase in length of this segment, the source of the additional cells (whether from propagation of the small population observed earlier near the macula densa or from differentiation of epithelial cells previously displaying diffuse COX-2-ir) was not discernible. After 1-2 wk at maximum strength, the numbers of COX-2 cells gradually declined over 2-3 wk to the minimal complement (averaging fewer than 2 per nephron) of normal adults.

Although the complement of COX-2 cells appeared to be slightly attenuated in some of the outermost (youngest) nephrons, all developing nephrons progressed through the COX-2 expression pattern described above. Thus the complement of COX-2 cells at P39 (Fig. 4, E and F) was still considerably greater than adult levels reached at approximately P60. An overview of COX-2 expression in kidney morphogenesis was gained by using BIOQUANT color image analysis to discriminate the intense mode of COX-2-ir and map its location in 4-µm thick sections of kidneys from a developmental series. Representative maps (Fig. 5) of the raw data illustrate the increase in size and number of COX-2 loci from birth to P7. From P7 through P28, the thickness of the cortical rim occupied by COX-2 cells remained rather constant even though the cortical thickness increased approximately threefold. This pattern reflected the balance between upregulation of COX-2 as the peripheral nephrons were generated and downregulation of COX-2 as the inner nephrons reached maturity.


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Fig. 5.   Topographic maps of COX-2 immunoreactivity in kidneys from rats staged from birth (P0) to adult. Magnifications are equivalent at all stages.

However, over this period, the number and sizes of the COX-2 loci were not altered significantly. Thus, as the perimeter of the kidney grew, the COX-2 loci appeared to become more dispersed. This subjective impression was confirmed by quantification of the percentage of cortical area occupied by COX-2-ir at each stage (Fig. 6A). After the peak at P7, the COX-2 percentage showed a steady decline to very low levels in adults. The total COX-2-ir per kidney was estimated by transformation of the two-dimensional area/area data into the third dimension using geometric models of cortex and medulla volumes (Fig. 6B). The volume occupied by COX-2 cells rose through P14 as new nephrons were generated, remained at high levels through P28, and declined as younger nephrons matured. These data correlate with the immunoblot data (Fig. 2) showing maximum immunoreactivity at P14; the blot signals at P21 and P28 reflect the constant volume of COX-2 being diluted in homogenates by increased kidney mass.


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Fig. 6.   A: area of COX-2-ir and area of cortex were measured for representative sections. Bars are means ± SD. B: volume of COX-2-ir per kidney was calculated using a geometric model of total cortical and medullary volumes.

In situ hybridization. COX-2 mRNA was localized in sections of GPAS-fixed kidneys using digoxigenin-labeled sense and antisense cRNA to the 3'-UTR of the rat COX-2 mRNA. With antisense probes, the blue alkaline phosphatase reaction product localized to cells of the TAL (Fig. 7, A and B); no staining was observed with the sense probe (not illustrated). At all ages examined from P0 to adult, the pattern of staining and cytological characteristics of cells positive for COX-2 mRNA strongly resembled the protein immunohistochemistry staining.


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Fig. 7.   A and B (high magnification of box in A): in situ hybridization. COX-2 mRNA is localized precisely to the specific regions of the TAL that immunostain for COX-2 protein. C and D (high magnification of box in C): papillary COX-2. At day P21, between the ducts (d) at the tip of the papilla, a subpopulation of interstitial cells exhibits COX-2-ir during adolescence. This expression is absent from normal adult kidney. Relative sizes of Fig. 3A scale bar: 500 µm (A), 125 µm (B, C), and 40 µm (D).

Papillary COX-2. Beyond the second postnatal week, a variable population of papillary interstitial cells displayed COX-2-ir (Fig. 7C). Although the COX-2-ir in these cells was quite dense, it appeared limited to the perinuclear region rather than permeating the cytoplasm as observed in the cortical COX-2 cells (Fig. 7D). A small number of these cells was usually present at the tip of the papilla in normal kidneys, and this population increased substantially in response to physiological renal stress, e.g., water deprivation (not illustrated). Even the most expanded populations, however, comprised only a fraction (50-60%) of the total interstitial cell population.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

The adult mammalian kidney is a particularly rich source for prostanoids, as well as an important biological target for these intrarenal prostaglandins. Prostaglandins regulate both renal hemodynamics and epithelial water and solute transport. In addition, there is evidence that fetal and early postnatal kidneys possess functional COX activity and are a rich source of prostaglandins. Fetal rat kidneys reportedly produce prostaglandins of the A, E, and F2 series (3). In postnatal rats, cortical PGE2 synthesis is highest in rats at 20 days of age compared with those aged 1 mo and 4 mo. In contrast, medullary PGE2 synthesis is lowest in the youngest rats and increases with age (21). Furthermore, PGE2 binding is fivefold higher in 5-day-old rat kidney compared with adult kidney (28).

Although previous studies have shown alterations in COX-2 expression in macrophages and other cells during inflammatory states (14, 16, 20, 29, 30), it also is present in kidneys of normal adults and during development. The present studies have localized COX-2 mRNA and immunoreactive protein in the kidneys of normal rats beginning at E16 and have demonstrated that the renal COX-2 expression is developmentally regulated. Northern and Western blots showed that COX-2 mRNA and protein are low at birth, rise to a peak in the first two postnatal weeks, and gradually decline to very low levels in normal adults. In situ hybridization and immunohistochemistry not only confirmed the blotting data but also demonstrated that the increased postnatal COX-2 signals were the result of expression at high levels in specific epithelial cells in the cortical segment of Henle's TAL.

During kidney development, COX-2-ir appears in three distinct forms. The earliest diffuse form is observed in midgestation embryonic stages, notably in cells undergoing induction and/or morphogenesis; this form is found in subcapsular epithelial structures in the kidney for the duration of nephrogenesis (through postnatal week 2 in the rat). The mature intense form of COX-2-ir appears primarily in functional nephrons as they mature; this is the form of COX-2 that is expressed under physiological regulation in the CTAL of adult kidney as described previously (9). The subcellular localization of intense COX-2-ir is of interest, because our images contrast with the customary localization of COX-2 to the endoplasmic reticulum (23). The intense COX-2-ir pervades even the most delicate profiles of the cell such as the basal interdigitations (Fig. 4D) that are packed with mitochondria and largely void of endoplasmic reticulum. Perhaps this form of COX-2 is free in the cytoplasm or associated with the plasmalemma. Similar sustained intense COX-2-ir is apparent in the distal vas deferens (unpublished observation). The third membrane-bounded form of COX-2-ir in the kidney is observed primarily in papillary interstitial cells, as well as in some transitional TAL cells during upregulation or downregulation of COX-2. The membrane-bounded COX-2 is restricted to the perinuclear region, probably within the nuclear envelope and cisternae of the endoplasmic reticulum. This form of COX-2 expression resembles that described previously for macrophages and cells in culture (23).

The timing of renal cortical COX-2 expression coincides with evidence suggesting that COX metabolites play important functional and developmental roles in the fetal kidney. Matson et al. (17) administered the nonspecific COX inhibitor, indomethacin, to fetal lambs during the third trimester of gestation. These lambs developed increased renal vascular resistance and decreased fetal renal blood flow, as well as increases in urinary sodium and chloride excretion and decreases in plasma renin activity, indicating that COX metabolites played important roles in the maintenance of fetal renal function. In humans an increased incidence of oligohydramnios was experienced by women who chronically consumed significant amounts of aspirin or other COX inhibitors during the third trimester of pregnancy (31). Since the fetal urine is the source of a significant amount of amniotic fluid, these studies suggested that inhibition of COX led to the suppression of fetal renal function.

There also is evidence that COX metabolites serve in mediation of normal renal development. Chronic administration of indomethacin to pregnant Rhesus monkeys led to renal hypoplasia in the neonates, with kidney mass reduced by 15% compared with control animals (26). The observed defect was specific for the kidney, since, in the treated animals, development of other organs was not affected, except for hepatic hypertrophy. Chronic use of COX inhibitors during human pregnancy has been related to fetal renal maldevelopment; kidneys from infants who came to term or died in the early postnatal period had few differentiated proximal tubules in the inner cortex and crowding of the glomeruli (34, 11). The outer cortex was more severely affected, with evidence of poorly differentiated glomeruli, undifferentiated tubule epithelia, and tubular dilation. In addition, the medullary pyramids were crowded with small immature tubules.

Metanephric cultures have provided evidence for potential roles of COX metabolites in nephrogenesis. In studies of growth requirements of E13 mouse metanephric explants grown in the absence of fetal calf serum, Avner et al. (1) determined that PGE1 was necessary for maximal growth and differentiation. Metanephric cultures not only showed an ~33% decrease in overall cellularity when grown in the absence of PGE1, but addition of PGE1 to the basal medium was absolutely necessary to induce metanephric differentiation (the formation of epithelial glomeruli). In the promotion of differentiation, PGE1 by itself was almost as effective as a complete hormonally defined medium containing selenium, insulin, transferrin, thyroxine, and PGE1.

Two recent reports of targeted disruption of murine COX-2 also have indicated an important role for this enzyme in renal development (5, 22). At maturity in homozygous (COX-2 -/-) animals, the kidneys were noted to be small, with a paucity of functional nephrons. Undeveloped mesenchymal tissue, immature glomeruli, and dysplastic tubules were present in the outer cortex. Hypoplasia or atrophy of the medulla accompanied by microcystic lesions in the corticomedullary junction also was observed in the knockout mice. The average life span of these mice was 3.5 mo, and they died of uremia. The pathological presentation appeared to be very similar to the above-noted descriptions of renal abnormalities in infants of mothers consuming large quantities of nonsteroidal antiinflammatory drugs. No apparent developmental or functional abnormalities have been described in mice with targeted disruption of COX-1 (13).

The placenta is known to be a rich source of prostaglandins; it has been demonstrated that in ovine placenta, COX-2 expression increases during the second half of pregnancy (7). Therefore, if prostaglandins or other COX metabolites play an important role in renal development, then it is possible that maternally produced products may contribute during pregnancy. In mice with targeted disruption of COX-2, the heterozygous mothers have apparently normal COX-2 expression and function, whereas the COX-2 -/- offspring have no COX-2 activity. It would be anticipated that these mice could help resolve the roles of maternal prostaglandins in fetal renal development. Indeed, one construct of the COX-2 -/- mice reportedly showed normal kidneys at birth, indicating that renal development may be promoted by maternal eicosanoids (22). However, COX-2 knockout on a different background produced renal anomalies in newborns (5). In both models, postnatal nephrogenesis was severely disrupted, as evidenced by dysgenesis of outer cortical nephrons.

Nephron genesis progresses centrifugally and in rodents continues through the second postnatal week. Therefore, the loci, time course, and magnitude of COX-2 expression demonstrated in the present study complement the COX-2 knockout data in pointing to sites where COX-2 plays critical roles in the development and maturation of nephrons. Although the precise nature of these roles is unclear, there is evidence that regulation of renin release by the juxtaglomerular apparatus (JGA) in fetal kidneys is similar to adult. Moutquin and Liggins (24) found that furosemide administration increased plasma renin activity in fetal sheep, indicating that the macula densa functions during fetal development, since it is known that furosemide-induced renin release is mediated by the macula densa (15). As mentioned above, inhibition of COX activity in fetal sheep reduced the levels of plasma renin (17). Our previous work demonstrating COX-2 cells in the TAL just proximal to the macula densa and increased numbers of COX-2 cells resulting from salt deprivation suggested that metabolic products of COX-2 may be signals in the tubuloglomerular feedback at the macula densa that regulates renin production (9). Experiments using an inhibitor specific to COX-2 have recently led to similar conclusions (8).

The large numbers of COX-2 cells in the perimacula densa region of developing nephrons would be consistent with hypotheses that higher levels of eicosanoids are necessary to drive the immature JGA or that they contribute signals leading to differentiation of the JGA. The recapitulation of COX-2 expression patterns in successive generations of nephrons strongly suggests that the COX-2 is involved in processes operating locally in the maturation of individual nephrons rather than in global physiological phenomena.

Finally, it may be hypothesized that regulation of COX-2 expression in the developing kidney involves glucocorticoids. It has been shown that glucocorticoids suppress expression of COX-2 (27). Because glucocorticoid levels in rodents decrease precipitously at parturition and remain very low for 2-3 wk (10), it is possible that this drop in ambient glucocorticoid levels promotes and sustains the increased expression of renal COX-2. Further studies will be necessary to understand the interrelations of COX-2 and corticosteroids in renal development.

In summary, COX-2 mRNA and immunoreactive protein, which are found at very low levels in normal adult rat kidney, are expressed at significant levels in a subset of the epithelial cells in the developing TAL as each nephron undergoes maturation. Intense COX-2-ir fills the cytoplasmic domain of cells that are concentrated in the vicinity of the nascent macula densa, but COX-2-ir is not observed in the macula densa cells themselves. As selective inhibitors of COX-2 become available, it will be important to assess their effects on renal morphogenesis.

    ACKNOWLEDGEMENTS

This work was performed in the George M. O'Brien Center for Kidney and Urologic Diseases supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-39261 and by funds from the Department of Veterans Affairs.

    FOOTNOTES

Address reprint requests to J. A. McKanna.

Received 24 April 1997; accepted in final form 7 August 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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