Increased expression of cyclooxygenase-1 and -2 in the diabetic rat renal medull - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:49 |只看该作者 |倒序浏览 |打印

作者：Rania Nasrallah, Anne Landry, Sonia Singh, Monika Sklepowicz,  Richard L. Hébert作者单位：Department of Cellular and Molecular Medicine and Kidney Research Centre, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5
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      【摘要】
   Alterations in renal prostaglandins (PGs) may contribute to some of the renal manifestations in diabetes leading to nephropathy. PG production is dependent on the activity of cyclooxygenases (COX-1 AND -2) and PG synthases. Our present study investigated levels of these enzymes in streptozotocin-diabetic rats at 2, 4, 6, and 8 wk of diabetes. Immunohistochemical analysis revealed an increase in COX signal in the inner and outer medulla of diabetic rats. This was confirmed by Western blotting, showing up to a fourfold increase in both COX isoforms at 4-6 wk of diabetes. Also, Western blot analysis revealed a sixfold increase in PGE 2 synthase expression in the outer medullary region of 6-wk diabetic rats but no difference in the inner medulla. In cultured rat inner medullary collecting duct (IMCD), levels of COX were increased two- to threefold in cells exposed for 4 days to 37.5 mM glucose compared with control of 17.5 mM. While no change in PGE 2 synthase levels was noted, PGE 2 synthesis was increased. Furthermore, levels of EP 1 and EP 4 mRNA were increased, as well as a twofold increase in EP 4 protein levels. Future studies will determine which COX isoform is contributing to the majority of PGE 2 produced in the diabetic IMCD and the significance of these findings to disturbances in IMCD function and to the progression of diabetic nephropathy.
      【关键词】 prostaglandin E streptozotocindiabetic rats inner medullary collecting duct EP receptor
   PROSTAGLANDINS ( PG s) ARE INVOLVED in diverse renal functions regulating hemodynamics and tubular transport processes. PGE 2 is by far the most prominent prostanoid (PG) produced in the kidney ( 3 ), particularly in the glomerular regions and the inner medulla. In part, the ubiquitous involvement of PGs in renal function is dependent on distinct G protein-coupled receptors, each one having a greater affinity for a respective prostanoid. For instance, PGE 2 elicits cellular responses by binding at least four EP receptor subtypes, EP 1-4 ( 8, 31 ). The past decade has led to many advances in the study of these receptors and the cellular responses linked to each one, especially in the kidney. Many investigators have localized each subtype to specific cell types all along the nephron ( 4, 29, 45, 46 ), and more recently nuclear localization of these receptors has been documented ( 1, 2 ). In addition, more and more insight is now available with respect the involvement of PGs and their receptors in certain renal pathologies ( 12, 25, 35 ). However, it is becoming quite evident that under normal conditions, PGs mainly participate in homeostatic functions, therefore serving to antagonize or enhance the cellular responses to other factors, such as ANG II ( 19, 40, 42 ). Thus it is conceivable that a disturbance in PG production and signaling will alter this balance and perpetuate a disease state.& w/ U& X  [) N9 f" s" `5 g- s4 b

The medullary region is an important contributor to the overall pool of renal prostanoids, in particular the inner medullary collecting ducts (IMCD). Renal PGE 2 production is dependent on the activity of two enzymes: cyclooxygenases (COX-1 and -2) and PGE 2 synthase ( 44, 49 ). Although it is believed that COX-1 is a constitutively expressed form of the enzyme and COX-2 is the inducible form ( 10 ), this has been proven untrue in the kidney. For instance, studies in the M-1 mouse cortical collecting duct cell line have shown that the intercalated cells of the collecting duct constitutively express both COX isoforms and that COX-2 contributes to the majority of the PGE 2 produced in these cells ( 11 ). Similarly, it has been noted that while COX-1 is present in collecting ducts, interstitial cells, and endothelial cells ( 24, 43 ), COX-2 is constitutively found in the macula densa cells, cortical thick ascending limb, medullary interstitial cells, and IMCD ( 16 ).
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Insights into the importance of each COX isoform came about by the generation of isoform-specific-deficient mice. While no major renal pathology was documented for the COX-1 knockout mice ( 26 ), COX-2-lacking mice display abnormalities in renal development and severe nephropathy ( 30 ). It is only when other underlying conditions exist that the significance of COX becomes recognizable. For example, after dehydration, COX-dependent PG production becomes an important survival mechanism in renal medullary interstitial cells ( 15 ). Thus it is notable to look at both enzymes as contributing to separate pools of PGs, and, depending on the cell type, the balance between cytoprotection and damage will determine the outcome and contribution to renal diseases.
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Diabetic nephropathy is a leading cause of end-stage renal disease. While alterations in PG levels have been implicated in the pathogenesis of diabetic nephropathy ( 20, 23, 27 ), resulting in hemodynamic changes and structural variations ( 9 ), the contribution of individual COX isoforms needs further clarification. Thus the purpose of this work is to study the expression of COX-1 and COX-2 in the outer and inner medulla of the kidney at 2, 4, 6, and 8 wk in streptozotocin (STZ)-diabetic rats by immunohistochemical analysis, Western blotting, and Northern blot analysis and to determine whether PGE 2 synthase levels are altered in STZ-diabetic rats. Finally, because PGE 2 is a key regulator of the natriuretic and diuretic functions of the kidney collecting ducts, the second part of this work will focus on changes in cultured rat IMCDs exposed to high glucose, studying PGE 2 levels and EP receptor expression.( a  K2 ?& |6 K8 Y6 v7 Z. w

MATERIALS AND METHODS- c, Q, r: A0 P5 Z9 P
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Diabetic rat model. Tissue from different kidney regions (cortex, outer medulla, inner medulla) of adult male Sprague-Dawley rats weighing 200-300 g was isolated under bright light using a dissecting microscope. The diabetic model utilized in the studies is STZ-diabetes, a widely used model resembling type 1 diabetes mellitus. STZ is an N -nitroso derivative of D -glucosamine that is utilized to induce diabetes in a variety of experimental animals and to help determine the long-term complications of diabetes. It basically functions as a toxin that selectively destroys the insulin-producing cells of the pancreas, rendering the rat diabetic within 24 h of injection ( 41 ). The animal care facilities at our disposal have a current protocol for inducing diabetes and maintaining the rats. Three different groups were employed: 1 ) vehicle-treated control rats (administered 0.1 M sodium citrate buffer, pH 4.0); 2 ) STZ-diabetic rats [administered 65 mg/kg STZ (Sigma, St.Louis, MO) in 0.1 M sodium citrate buffer, pH 4.0; hyperglycemia was maintained between 11 and 17 mM with daily subcutaneous injections of 1-2 U of insulin]; and 3 ) STZ-insulin rats (same as group 2 except for maintenance of euglycemia by subcutaneous implantation of a sustained-release insulin implant; Linplant, Linshin, Scarborough, ON). The day after STZ administration, urinalysis was performed for glucose and ketones using a Keto-Diastix reagent strip (Bayer, Etobicoke, 111 mM, the animal automatically received 1.5 U of insulin. However, if the urinary glucose was at 56 mM, a blood glucose test was performed, and when levels were 27-44 mM the animal was given a dose of insulin. Otherwise, blood glucose was not tested daily. Animals with sustained glucosuria were assigned to STZ-diabetic or insulin groups. Throughout the study, 1.5-2 U of insulin were sufficient to maintain blood glucose levels between 10 and 17 mM. Experiments were performed in rats in early stages of diabetes at 2, 4, 6, and 8 wk after STZ injections as well as in matched controls for each stage. Our project follows the guidelines from the Canadian Council on Animal Care and meets the ethical guidelines for our institution. The body weights of each animal were recorded daily, and kidney weights were measured once the animal was killed. These data are summarized in Table 1.
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Table 1. Summary of body weights and kidney weights for rats at different stages of STZ-diabetes5 C# i4 k. M3 ]4 a6 ]1 w1 a+ N

Immunohistochemistry. Kidneys were removed from control, diabetic, and insulin-treated rats at 2, 4, 6, and 8 wk after STZ injection and fixed in 4% paraformaldehyde/0.2% picric acid in PBS for 18 h at 4°C. Paraffin-embedded longitudinal sections 4 µm thick were then permebealized for 15 min in 0.3% Triton X-100/PBS and incubated with COX-1 or COX-2 polyclonal antibodies (Cayman Chemical, Ann Arbor, MI) for 18 h at 4°C. After incubation with biotinylated anti-rabbit IgG for 30 min at 37°C, the sections were incubated with streptavidin-linked horseradish peroxidase, and diaminobenzidine substrate was used to visualize the signals. Counterstaining was performed with Mayer's hematoxylin, and sections were analyzed using a Zeiss microscope.3 Q8 J& d) m9 g+ ?9 k- H

IMCD cell culture. The inner medullary regions from five rats were pooled and minced on ice in a petri dish. IMCDs were isolated as previously described ( 33 ) by bubbling in 5% CO 2 -air at 37°C in a solution of collagenase/DNase, followed by osmotic shock. IMCD cells were cultured in DMEM-F-12 containing 10% ( day 1 ), then 2% FBS, 1% P/S, 5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml selenium, 2.5 nM triiodothyronine/sodium salt, and 50 nM hydrocortisone (Sigma). Cells were grown at 37°C and 5% CO 2 in media containing 1 ) control (17.5 mM glucose in DMEM-F-12), 2 ) 30 or 37.5 mM glucose, or 3 ) 30 or 37.5 mM mannitol (osmolarity control). The glucose in the media remained constantly above 35 mM until 48 h after initial plating, when it dropped to 25-30 mM (measured using Keto-Diastix glucose indicators). Therefore, the culture media was changed at 2 days to ensure exposure of cells to 37.5 mM glucose over the 4-day period. After 3 days, the cells were serum starved in similar media for 24 h before the start of the experiments.5 J0 s7 ]9 t) H% [' N" O8 Z$ X
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Western blotting. Protein lysates from the outer and inner medulla were obtained by homogenizing the tissue in a 25 mM Tris · HCl lysis buffer. For IMCD cell cultures, protein samples were prepared by lysing cells in 100 mM Tris · HCl (pH 7.4) containing 1 mM EDTA and 1 mM EGTA, followed by sonication for 5 s using an Ultrasonics cell disrupter. The cell lysates were then centrifuged at 10,000 g for 10 min, and the supernatant was removed. Twenty-five micrograms of each sample were resolved by SDS-PAGE on a polyacrylamide gel and transferred to a nitrocellulose membrane. After blocking O/N in 5% milk/TBS-T, the membrane was incubated with either anti-COX-1 or anti-COX-2 polyclonal antibody. After incubation with a horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody, ECL was used to visualize the signals. A single band of 70 or 72 kDa was obtained for COX-1 and COX-2, respectively. The samples were then normalized with detection of -actin, and a densitometric analysis was performed. Tables 2 and 3 summarize the data obtained at 2, 4, 6, and 8 wk of STZ-diabetes. PGE 2 synthase levels were examined in outer and inner medullary samples at 6 wk of diabetes and in cultured IMCD treated with either glucose or mannitol, using an anti-PGE 2 synthase antibody (Cayman Chemical) detecting a 16-kDa product. Also in cultured IMCD, the EP 4 receptor was detected using a human polyclonal -EP 4 IgG (Cayman Chemical) diluted 1:5,000 in 10% milk in TBS-T.6 b* X! }2 K+ S0 c9 V

Table 2. Summary of COX-1 levels at different stages of STZ-diabetes

Table 3. Summary of COX-2 levels at different stages of STZ-diabetes

Northern blotting. Kidneys were removed from control, diabetic, and insulin-treated rats at 6 wk after STZ injection. Total RNA was isolated from samples of outer and inner medulla using the TRIzol method as described by the manufacturer (GIBCO-BRL) and was DNase treated (Boehringer Mannheim) to eliminate genomic DNA. Ten micrograms of total RNA from each sample were loaded onto a formaldehyde gel. RNA was then transferred to a nitrocellulose membrane. After baking for 2 h (80°C, vacuum), the membrane was incubated O/N with a [ 32 P]dCTP-labeled human COX-2 cDNA probe, then exposed to film for 1 wk. To normalize the samples, the expression of -actin was determined for the same membrane by reprobing with a human anti- -actin cDNA (Cayman Chemical) after the membranes were stripped in boiling 0.5% SDS. IMCD cells were grown to confluence in 100-mm petri dishes, scraped off, and then centrifuged at 1,100 rpm. The collected pellet was resuspended in 1 ml TRIzol reagent (GIBCO), and total RNA was isolated using the TRIzol method as described by the manufacturer and DNase treated (Boehringer Mannheim) to eliminate genomic DNA. Densitometric analysis was used to compare the relative expression of COX-2 in freshly isolated medullary tissue and of EP 1 and EP 4 receptor mRNA in each IMCD sample using mouse EP 1 and EP 4 cDNA probes (a gift from Dr. Matthew Breyer, Vanderbilt Univ.). Data are presented as means (fold of control) ± SE., [0 |; }7 c0 \+ `) S

Enzyme immunoassays. Cultured rat IMCDs were exposed to either 37.5 mM glucose or mannitol, and PGE 2 levels in these samples were compared with control (17.5 mM glucose). To quantify the amount of each prostanoid (PGE 2, 6-keto-PGF 1 ) being produced, the supernatant was removed from each culture dish, and competitive enzyme immunoassays (EIA; Cayman Chemical) were performed. Production of prostanoids in IMCD at 2 and 4 days was analyzed by EIA according to the manufacturer's instructions. Briefly, the assay is based on a competitive binding of PGE 2 or 6-keto-PGF 1 and their respective acetylcholinesterase conjugate (tracer) for a limited amount of monoclonal antibody. Because the tracer concentration is held constant, the amount of tracer bound to the antibody will be inversely proportional to the amount of PG in the sample. Detection is based on a colorimetric reaction using Ellman's reagent, which contains the substrate to acetylcholinesterase. The intensity is then determined by spectrophotometry.' ]. l) p1 g/ j- F

Statistics. SigmaPlot software for Windows, version 4.01 (1986-1997), was used to analyze data. Results are expressed as means ± SE. A one-way ANOVA was performed to assess the statistical significance between data points, followed by Tukey's test for comparison of values.# c. b9 y) @. a! C% a: ~" m

RESULTS& N' ?, Q, v1 N: ^3 @

COX-1 and COX-2 are increased in the STZ-diabetic rat medulla. Immunohistochemical analysis of COX-1 and COX-2 revealed that both are present in tubular segments of the outer (outer medullary collecting ducts, medullary thick ascending limb) and inner (IMCDs) medulla in control rats. Although there were no obvious changes in distribution of COX isoforms throughout the medullary regions on injection of STZ, there was clearly an increase in the staining intensity at 4 and 6 wk of STZ-diabetes (Figs. 1 and 2 ). This increased intensity is reversed in the insulin-treated group. Previous experiments in our laboratory confirmed that each antibody is specific for its respective COX isoform ( 11 ). As indicated in Fig. 3, Western blot analysis confirms the immunohistochemical findings, showing a two- to fourfold increase in COX-1 between 2 and 6 wk of STZ-diabetes and about a threefold increase in COX-2 at 6 wk ( Fig. 4 ). A summary of the levels of COX-1 and -2 at different stages of diabetes is shown in Tables 2 and 3. In contrast to changes in protein levels, Northern blotting indicates that COX-1 and -2 mRNA is unchanged in the medullary region of 6-wk STZ-diabetic rats (data not shown).
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Fig. 1. Immunohistochemical analysis of cyclooxygenase (COX)-1 at 4 wk. Longitudinal sections of paraffin-embedded kidneys were used to localize COX-1 in the medullary regions of the kidney of 4-wk streptozotocin (STZ)-diabetic rats compared with controls. COX-1 was detected using a human polyclonal antibody. Increased tubular staining is shown by arrows in diabetic sections. A - C : outer medulla. D - F : inner medulla. A and D : control. B and E : diabetic. C and F : insulin ( n = 3).
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Fig. 2. Immunohistochemical analysis of COX-2 at 6 wk. Longitudinal sections of paraffin-embedded kidneys were used to localize COX-2 in the medullary regions of the kidney of 6-wk STZ-diabetic rats compared with controls. COX-2 was detected using a human polyclonal antibody. Increased tubular staining is shown by arrows in diabetic sections. A - C : outer medulla. D - F : inner medulla. A and D : control. B and E : diabetic. C and F : insulin ( n = 3).
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Fig. 3. COX-1 protein is increased at 6 wk of diabetes. A : representative autoradiograph of COX-1 (70 kDa) in outer medulla. Lanes ( left to right ): control, diabetic, insulin. B : detection of -actin to normalize samples. C : densitometric analysis. Results shown as fold of control. Values are means ± SE; n = 6. * P

Fig. 4. COX-2 protein is increased at 6 wk of diabetes. A : representative autoradiograph of COX-2 (72 kDa) in outer medulla. Lanes ( left to right ): control, diabetic, insulin. B : detection of -actin to normalize samples. C : densitometric analysis. Results are shown as fold of control. Values are means ± SE; n = 5. * P. T+ p& B+ D' i
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PGE 2 synthase levels are increased in outer medulla of 6-wk STZ-diabetic rats. A secondary enzyme in the synthesis pathway of PGE 2 is PGE 2 synthase, which converts the inactive intermediate PGH 2 produced by the action of COX on arachidonic acid. Western blotting was utilized to measure protein levels of this enzyme in diabetic rats compared with controls. A 5.9 ± 1.9-fold increase in PGE 2 synthase protein was observed in the outer medulla of 6-wk STZ-diabetic rats ( Fig. 5 ), but no change in enzyme levels was found in the inner medulla. Consistent with this latter finding, the levels of PGE 2 synthase were unchanged (control 1.1 ± 0.2-fold, n = 4) in cultured rat IMCD exposed to 37.5 mM glucose for 4 days compared with 17.5 mM for controls.
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Fig. 5. PGE 2 synthase is increased in outer medulla of STZ-diabetic rats. Protein lysates were obtained from medullary regions of rats at 6 wk. A : representative autoradiograph of PGE 2 synthase (16 kDa) in outer medulla. Lanes ( left to right ): control, diabetic, insulin. B : detection of -actin to normalize samples. C : densitometric analysis of PGE 2 synthase in outer and inner medulla. Results are shown as fold of control. Values are means ± SE; n = 5-6. * P
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COX-1 and COX-2 are increased in IMCD on exposure to high glucose. To determine whether the increase in COX observed in the medulla of 4- to 6-wk STZ-diabetic rats also occurred in the IMCD exposed to high glucose, we cultured IMCD cells in 37.5 mM glucose for 4 days. As illustrated in Fig. 6, COX-1 and COX-2 levels were augmented 2.1 ± 0.4- and 3.1 ± 0.4-fold, respectively, in high-glucose-treated cells compared with controls. As noted above, for 6-wk STZ-diabetic rats no change in COX-1 and -2 mRNA was noted by Northern blotting in cultured rat IMCD exposed to high glucose (data not shown).
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Fig. 6. COX-1 and -2 are increased in cultured rat inner medullary collecting duct (IMCD) exposed to high glucose. Protein was isolated from IMCD exposed to control, 37.5 mM glucose, or 37.5 mM mannitol for 4 days and quantified by Western blotting. Results of densitometric analysis of COX are shown as fold of control. Values are means ± SE; n = 3-4. * P
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PGE 2 synthesis is increased in IMCD on exposure to high glucose. Despite the lack of change in PGE 2 synthase levels in the inner medulla and IMCD (as noted above), a significant increase in COX was observed. Because COX is the rate-limiting enzyme in the PGE 2 synthesis pathway, we measured PGE 2 levels by enzyme immunoassay. As shown in Fig. 7, PGE 2 levels are elevated 2.05 ± 0.46-fold in IMCD exposed to high glucose for 4 days, but no change in 6-keto-PGF 1 levels was observed. In comparison, the levels of both prostanoids were elevated to a similar extent after 2 days of high-glucose exposure: 1.7 ± 0.3-fold control for PGE 2 and 1.6 ± 0.17-fold control for 6-keto-PGF 1. It is noteworthy that thromboxin X 2 levels were also measured, but they did not differ from control (data not shown).7 Y; K( W: f* g/ B8 E) a0 K' N5 Q/ c
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Fig. 7. Prostanoid levels are increased in cultured rat IMCD exposed to high glucose. IMCD were exposed to control, 37.5 mM glucose, or 37.5 mM mannitol for 2 ( A ) and 4 days ( B ). Production of PGE 2 and 6-keto-PGF 1 was assayed by competitive enzyme immunoassay, showing results as fold of control. Values are means ± SE; n = 6.* P P* A) N+ K! `; ?1 k1 c# }) f& L

EP 1 and EP 4 receptors are altered in IMCD on exposure to high glucose. As illustrated in Fig. 8, both EP 1 and EP 4 receptor mRNAs are detected in rat IMCD. This medullary localization of EP receptor subtypes is in agreement with our previous work demonstrating the expression of EP 1, 3, 4 receptor mRNAs in medullary segments of the rat nephron, namely EP 1, 3 in medullary thick ascending limb ( 18, 32 ) and EP 1, 3, 4 in IMCD ( 33 ). As shown in this study, on exposure of cells to high glucose, there is a significant increase of 1.8-fold in EP 4 receptors. Interestingly, when mannitol was added instead of glucose there is a slight reduction in EP 4 mRNA. While a tendency for EP 1 mRNA to increase is noted, the relevance remains inconclusive due to an increase in response to mannitol as well. Whether the change in mRNA is due to an osmotic effect is noteworthy, and ongoing work in our laboratory will further examine this different effect on both receptors. Furthermore, Western blot analysis indicates that EP 4 receptors are elevated in IMCD cultured in 37.5 mM glucose about twofold compared with control cells grown in 17.5 mM glucose ( Fig. 9 ). This is consistent with previous reports from our laboratory indicating that a change in cortical collecting duct COX levels resulted in compensatory changes in PGE 2 receptor levels at the cell surface ( 32 ).+ e5 P' P- R4 y8 x! f9 U$ D9 r

Fig. 8. EP receptor mRNA is increased in IMCD exposed to high glucose. Northern blot detection of EP receptor mRNA in cultured IMCD exposed to control, 37.5 mM glucose, or 37.5 mM mannitol for 4 days is shown. Densitometric analysis of EP 1 and EP 4 receptor mRNA levels are shown as fold of control. Values are means ± SE; n = 3. * P P$ u: o% a* a- O1 _) Z- {

Fig. 9. EP 4 receptors are increased in cultured rat IMCD exposed to high glucose. IMCD were exposed to control, 37.5 mM glucose, or 37.5 mM mannitol. EP 4 receptors are presented as fold of control. Values are means ± SE ( n = 6). * P8 y% J# }: t9 p7 J' J

DISCUSSION. b+ x' m- [& V6 d9 y! P3 N5 K
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Numerous studies allude to the putative role of prostanoids (PGs) in diabetic nephropathy ( 9 ), but a major controversy exists as to the nature of their involvement: whether they propagate the complications or serve to antagonize the deleterious effects of other agents. Moreover, present work has examined the beneficial effects of specifically targeting certain prostanoid pathways to alleviate the manifestations of the disease, including EP 1 receptor antagonists ( 28 ) and IP receptor agonists ( 22, 48 ). However, the underlying mechanisms of PG involvement remain uncertain. In diabetic kidneys, it has been clearly demonstrated that COX enzymes are elevated, and glomerular PG production in most species is increased ( 13, 20, 47 ) as well in STZ-diabetic rats ( 25, 39 ). Also, several pathways are stimulated by hyperglycemia and are putatively responsible for altering the levels of COX, including activation of protein kinase C ( 36 ), p38 mitogen-activated protein kinase ( 7, 14 ), and countless others ( 21, 34 ). Actually, selective inhibitors of COX-2 (NS-398) have been used to reverse some of the renal complications of STZ-diabetes, such as altered glomerular filtration rate, without affecting mean arterial pressure or renal plasma flow ( 23 ). Also, in the remnant kidney model, COX-2 inhibition slowed the development of proteinuria and attenuated renal structural damage in animals treated with these drugs ( 38 ). The exact mechanisms by which COX-derived prostanoids participate in the pathogenesis of nephropathy, and the extent of the contribution of individual COX isoforms, surely require further elucidation. However, it is clearly no longer valid to label COX-1 products as beneficial for normal function and COX-2-derived prostanoids as playing a pathological role.
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In this study, we characterized the medullary expression of COX-1 and COX-2 in diabetic rats. Tubular levels (IMCD, outer medullary collect duct, medullary thick ascending limb) of both COX isoforms are elevated between 4 and 6 wk after STZ injection, as well as in cultured IMCD exposed to high glucose for 4 days. This is in agreement with a previous study by Komers et al. ( 23 ) showing increased cortical expression of COX-2 at 4 wk of diabetes. However, our study clarifies the time course for each COX isoform, between 2 and 8 wk of diabetes. Because we did not find a change in COX mRNA in either the diabetic rats or in IMCD exposed to high glucose, it is possible that there is increased protein stabilization; however, whether there is also enhanced enzyme activity is uncertain. The renal manifestations, or cellular events, coinciding with this increase, and their role in the pathogenesis of the nephropathy at these stages of diabetes, remain unclear at this time.

Another important enzyme in PGE 2 synthesis is PGE 2 synthase. While COX is increased in both the inner and outer medulla of the diabetic kidney, changes in PGE 2 synthase were only observed in the outer medulla. Consistent with this finding, we show no change in this enzyme in cultured IMCD exposed to high glucose. Thus the contribution of PGE 2 synthase to diabetic alterations in PG levels in the inner medulla seems insignificant. To verify whether the increase in COX in IMCD exposed to high glucose resulted in changes in prostanoid levels, despite the lack of change in PGE 2 synthase, we measured the production of PGE 2 and prostacyclin in the media; these are the two major prostanoids produced in the rat inner medulla ( 3 ). Our work indicates an increase in both PGE 2 and prostacyclin (6-keto-PGF 1 ) in IMCD exposed to high glucose for 2 days and an increase in only PGE 2 at 4 days. However, at this time it is not clear which COX isoform contributes to the majority of PG production in the rat IMCD. In a previous study by our group ( 11 ), it was demonstrated that in a mouse cortical collecting duct cell line, the majority of PGE 2 produced was dependent on COX-2 rather than COX-1. Future studies using NS-398 (a selective COX-2 inhibitor) will determine whether this holds true in the rat IMCD as well.
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To further characterize changes in the IMCD exposed to high glucose, we examined the expression of PGE 2 receptors in these cells. We show by Northern blot analysis that both EP 1 and EP 4 receptor mRNAs are present in cultured rat IMCD, as previously observed by RT-PCR in our laboratory ( 33 ). While exposure to high glucose increased the expression of EP 1, the response was similar on exposure to mannitol, indicating a possible osmotic effect. Ongoing studies in our laboratory will address this issue. On the other hand, EP 4 mRNA was significantly increased, as well as EP 4 protein levels in the IMCD exposed to high glucose. It is noteworthy that work looking at the effects of high glucose on the PGE 2 response in primary cultures of glomerular mesangial cells showed an inhibition of PGE 2 -stimulated cAMP, with no change in EP 4 receptor mRNA ( 20 ). The reason for the discrepancy in regulation of EP 4 mRNA by glucose in IMCD vs. mesangial glomerular cells is unclear at this time. Future studies in our laboratory will examine the cellular signaling in response to PGE 2 to clarify the effects of glucose in the IMCD. For instance, increases in cAMP are known to inhibit the proliferative state of cells ( 6, 20 ), determine cell fate and apoptotic responses ( 17 ), and alter gene transcription via cAMP-responsive elements in a large number of target genes ( 34 ).
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As observed in this study, rather than making up for increased PGE 2 production by downregulating EP 4 receptors, IMCD cells exposed to high glucose have an enhanced cellular response, by increasing both COX (PGE 2 production) and cell surface binding sites for PGE 2. Whether this would result in a change of IMCD function that could perpetuate tubular disease in diabetics requires further investigation. The IMCD is an important regulator of sodium, H 2 O, and potassium homeostasis, disturbances of which are three of many seen in diabetes. Because PGE 2 is a key mediator of this IMCD function, acting through at least three subtypes of the EP receptors, EP 1, EP 3, and EP 4 ( 5, 46 ), defective PGE 2 /EP receptor signaling pathways could interfere with the fine-tuning of salt and water transport, and these abnormalities could contribute to edema, hypertension, and vascular changes associated with diabetic nephropathy. However, we report in this study that tubular PGE 2 levels are increased in our high-glucose diabetic model. Because PGE 2 plays an important role in regulating ion concentrations in the urine by inhibiting NaCl transport in the collecting duct, favoring salt elimination in the urine together with water ( 5 ), the resultant natriuresis and diuresis may be beneficial in diabetes as a compensatory response. To further support this idea, the significance of PGE 2 to the maintenance of salt and water homeostasis is clearly demonstrated by the undesirable renal effects such as sodium and potassium retention ( 37 ), associated with the use of nonsteroidal anti-inflammatory drugs, which inhibit the production of PGEs. It would be very interesting if fluctuations in PGE 2 /EP signaling could be linked in time to the state of the rat in vivo, showing that, at various stages of the disease, levels are increased or decreased according to factors such as glomerular filtration rate, blood pressure, etc. Therefore, we report here that the PGE 2 system in the IMCD is playing a protective role, compensating for systemic disturbances that are associated with the disease. Adding to the complexity, PGE 2 can also play a role within the IMCD itself, independent of H 2 O and electrolyte transport. It could potentially alter the expression of numerous genes via direct nuclear signaling pathways ( 1, 2 ) and thus contribute to nephropathy by cross talking with nitric oxide or ANG II, for instance. It could also alter fibrogenesis or apoptotic events that are associated with tubular atrophy and cell loss in later stages of diabetes. The possibilities are endless, and needless to say more work is required to shed some light on the mechanisms involved., L  D' x2 Q! b/ |) c9 d

In summary, immunohistochemical analysis reveals more intense COX-1 signals in tubule segments of outer and inner medulla at 4 wk of diabetes; COX-2 staining is stronger at 6 wk. COX-1 protein levels are also increased at 2-6 wk of diabetes, but COX-2 is only increased at 6 wk. No change in COX mRNA is detectable. A sixfold increase in PGE 2 synthase is observed in the outer medulla at 6 wk of diabetes, but no difference is seen in the inner medulla or IMCD. On exposure to high glucose, synthesis of PGE 2 and prostacyclin is increased in the IMCD. Similarly, EP 4 receptor mRNA and protein are increased in IMCD exposed to high glucose. Further studies will clarify which isoform of COX is contributing to increased PGE 2 in the diabetic IMCD and the significance of these findings to disturbances in IMCD function and progression of diabetic nephropathy. Once clarified, this could lead to the advent of better combination therapy to prevent the progression of the disease., w* `5 n# y% g" X! F

DISCLOSURES1 Z. Z0 k( T1 F1 O, b/ [

This work was supported by the Kidney Foundation of Canada and the Canadian Institutes for Health Research (MT-14103).+ T6 Y% H* p/ \; O+ y. r* ^

ACKNOWLEDGMENTS  T' `) D1 w6 P6 G

We thank Dr. Matthew D. Breyer (Vanderbilt Univ., Nashville TN) for kindly providing us with mouse EP receptor probes." E* g, L# k% |1 }3 x7 C2 H% `
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