Differential regulation of COX-2 expression in the kidney by lipopolysaccharide: role of CD14

Tianxin Yang1, Daqing Sun2, Yuning G. Huang1, Ann Smart1, Josephine P. Briggs1,2,3, and Jurgen B. Schnermann2

Departments of 1 Internal Medicine and 2 Physiology, University of Michigan, Ann Arbor, Michigan 48104; and 3 National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892


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Induction of the inducible cyclooxygenase isoform COX-2 is likely to be an important mechanism for increased prostaglandin production in renal inflammation. We examined the effect of lipopolysaccharide (LPS) on regional renal COX-2 expression in the rat. In the inner medulla, LPS injection (4 mg/kg ip) induced a twofold and 2.5-fold increase in the levels of COX-2 mRNA and COX-2 protein, respectively. In contrast, COX-2 expression in the renal cortex was not significantly altered. COX-2 promoter transgenic mice were created using the 2.7-kb flanking region of the rat COX-2 gene. In these animals, LPS injection induced reporter gene expression predominately in the inner medulla. The LPS receptor CD14, usually regarded as a monocyte/macrophage-specific marker, was found to be abundantly expressed in the inner medulla and in dissected inner medullary collecting duct (IMCD) cells, suggesting that it may mediate medullary COX-2 induction. CD14 was present only at low levels in cortex and cortical segments, including glomeruli. In cultured cells, it was abundant in mouse IMCD (mIMCD-K2) cells and renal medullary interstitial cells, but largely undetectable in mesangial cells and M1 cells, a cell line derived from mouse cortical collecting ducts. In the mIMCD-K2 cell line, LPS significantly induced COX-2 mRNA expression, with concomitant induction of CD14. LPS-stimulated COX-2 expression was reduced by the addition of an anti-CD14 monoclonal antibody to the culture medium. These results demonstrate that LPS selectively stimulates COX-2 expression in the renal inner medulla through a CD14-dependent mechanism.

cyclooxygenase-2; renal inner medulla; transgenic mice


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CYCLOOXYGENASE (COX; prostaglandin-endoperoxide synthase EC 1.14.99.1) catalyzes the oxygenation and peroxidation of arachidonic acid to generate prostaglandin endoperoxides, the immediate precursors of a series of bioactive prostaglandins. Two distinct isozymes of COX have been identified, COX-1 and COX-2 (7, 9, 25, 27). COX-1 is constitutively expressed in a wide variety of tissues and organs, and is generally believed to have "housekeeping functions," especially in stomach, spleen, and kidney. Expression of COX-2, in contrast, can be rapidly induced, most dramatically in inflammatory cells, by proinflammatory stimuli such as cytokines, mitogens, and lipopolysaccharide (LPS). Selective inhibition of COX-2 has been shown to reverse inflammation in various tissues (1). Recently, large-scale clinical trials have demonstrated that COX-2 inhibitors are as potent as nonselective COX inhibitors, such as indomethacin and ibuprofen, in the treatment of various inflammatory diseases, but that they have fewer side effects in stomach and platelets (11, 23, 26). These observations are consistent with the initial speculation that COX-2 is the cytokine-inducible cyclooxygenase, critically involved in inflammatory responses (7, 16).

There is substantial evidence to demonstrate that LPS induces unique inflammatory responses in the kidney. Systemic infusion of LPS causes marked renal vasoconstriction, leading to a marked reduction of renal blood flow, despite a normal or increased cardiac output (2, 18). LPS also suppresses renin release (4, 28) and is associated with an immediate reduction of Na+ and K+ excretion followed by the development of profound natriuresis (19). It appears that local mechanisms underlie LPS-induced alterations in renal function. Prostaglandins are produced abundantly in the kidney, particularly in the inner medulla, and are capable of modulating multiple renal functions, such as renal blood flow (5, 34), renin release (33), and urinary Na+ excretion (3). LPS, infused systemically or administered intrarenally via the renal artery, has been shown to stimulate prostaglandin release from the kidney (29). These observations are consistent with the hypothesis that prostaglandins may be involved in the LPS-induced renal pathophysiology.

The mechanism of action of LPS has been extensively studied. LPS first induces hepatic LPS binding protein (LBP), which functions as a carrier protein in the circulation. The complex of LPS and LBP then recognizes and acts on CD14, which is located on the surface of macrophages (31). CD14 is a 5300-5500 mol wt glycosyl-phosphatidylinositol-anchored membrane protein that is found almost exclusively in monocytes and macrophages and that is often used as a marker of monocyte lineage. Large numbers of studies demonstrate that CD14 binds to LPS and LBP, and mediates LPS-induced cytokine production (6, 37). CD14, therefore, functions as the receptor for the LPS/LBP complex. In support of this notion, transgenic mice expressing human CD14 are hypersensitive to LPS (10).

The current experiments were performed to determine 1) whether treatment with LPS affects COX-2 expression in the kidney and whether the induction of COX-2 by LPS differs between different regions of the kidney, 2) whether LPS affects COX-2 promoter activity in transgenic mice in vivo, 3) whether renal epithelial cells express CD14, and 4) whether CD14 plays a role in LPS-regulated COX-2 expression in renal cells.


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Animals. We performed experiments in male Sprague-Dawley rats (175-200 g). Rats in the experimental group (n = 16) received an intraperitoneal injection of 4 mg/kg Escherichia coli LPS (Sigma Chemical, St. Louis, MO). Rats in the control group (n = 16) received an intraperitoneal injection of the same volume of PBS. The animals were killed 2, 4, 6, and 8 h after LPS injection and tissue samples from renal cortex and inner medulla were harvested for the determination of COX-2 mRNA and protein. Animals were killed and tissue samples from renal cortex and inner medulla were harvested for the determination of COX-2 mRNA and protein. For RNA extraction, tissue samples were transferred immediately into microcentrifuge tubes containing 300 µl of TRI Reagent (Molecular Research Center, Cincinnati, OH) and snap frozen in liquid nitrogen. Samples from the other kidney were collected for protein analysis.

Cell culture experiments. The source of cultured cells, including mouse inner medullary collecting duct cells (mIMCD-K2), mouse cortical collecting duct cells (M1 cells), mesangial cells, and renal medullary interstitial cells (RMIC), has been described previously (39). To study the effect of LPS on COX-2 expression, we serum deprived confluent mIMCD-K2 cells for 12 h and then treated them with LPS (100 ng/ml) for various periods of time. To study the effect of CD14 receptor blockade on LPS-induced COX-2 expression, we serum deprived confluent mIMCD-K2 cells for 12 h and treated them with rmC5-3 (0.05 mg/ml; PharMingen, San Diego, CA), an antibody against recombinant mouse CD14, before stimulation with LPS.

cDNA synthesis and PCR. Details of the methods used have been published previously (39). The sequences of the oligonucleotide primers and their location in the published cDNA sequences were as follows: 1) COX-2: sense 5'-ACA CTC TAT CAC TGG CAT CC-3' (bp 1229-1248) and antisense 5'-GAA GGG ACA CCC TTT CAC AT-3' (bp 1794-1813) were predicted to amplify a product of 584 bp (9); 2) CD14: sense 5'-GCA GCA GTG GCT AAA GCC TG-3' (bp 882-901) and antisense 5'-ATC GCC TCT TTG TTT AAG GAA CAC-3' (bp 1185-1208) were predicted to amplify a product of 627 bp (12). We performed 30 PCR cycles with an annealing temperature of 59°C for both COX-2 and beta -actin PCR and 60°C for CD14 PCR. The quantitative assays used 10-fold serial dilutions of cDNA of each experimental sample to ensure that all PCR determinations fell within the linear amplification range. RNA samples subjected to the PCR procedure without prior reverse transcription served as "reverse transcriptase-minus" controls. Water and dissection medium blanks were run to serve as a control for cDNA contamination. We checked genomic DNA contamination by carrying samples through RNA isolation and cDNA synthesis without the addition of reverse transcriptase. Dissection medium and reverse transcriptase-negative samples consistently yielded no product.

Western blotting analysis. Immunoblotting was performed as previously described (35). Briefly, kidneys were removed and tissue samples from cortex (200 µg) and inner medulla (100 µg) were dissected and homogenized. Tissue homogenates were suspended in loading buffer and subjected to electrophoresis on 10% SDS-PAGE. We transferred the contents of the gels electrophoretically to nitrocellulose membranes and stained them briefly with Ponceau S to determine uniformity of electrophoretic transfer. The membranes were then washed in TBS (20 mmol/l Tris · HCl, pH 7.5; 150 mmol/l NaCl; and 1% Nonidet P-40), blocked with 5% dry milk in TBS, and incubated with a 1:500 dilution of a COX-2 polyclonal antiserum (Cayman). The membranes were washed three times in TBS, blocked again, and incubated with 125I goat anti-rabbit IgG (ICN Biomedicals, Irvine, CA). Finally, we washed the filters three times in TBS, dried them, and quantitated them by phosphorimage analysis using a GS-250 Molecular Imager System and Phosphor Analyst software (Bio-Rad, Hercules, CA).

Experiments in transgenic mice. We amplified 2.7 kb of previously characterized rat COX-2 promoter sequence from genomic DNA isolated from liver of Wistar-Kyoto rats using a high-fidelity amplification kit (Boehringer Mannheim). The fragment was subsequently subcloned into pnlacf vector upstream of the nuclear localization signal and lacZ gene. The recovery of fertilized eggs, injection, and reimplantation into the oviducts of pseudopregnant female mice were performed with assistance from the Transgenic Core Facility at the University of Michigan (20). We performed genotyping by PCR using the chimeric, construct-specific primers. We obtained F1 mice by breeding the founder mice with CD-1 mice. After two intraperitoneally injected doses of LPS (1 mg/kg each), at 12 and 2 h before death, animals were anesthetized and perfused through the heart with 4% paraformaldehyde. Kidney was removed and cut into pieces. X-gal staining was performed at 30°C for 20 min, with the solution containing 0.2% X-gal, 10 mM sodium phosphate buffer (pH 7.0), 150 mM NaCl, 1 mM MgCl2, 3.3 mM K4Fe(CN)63H2O, and 3.3 mM K3Fe(CN)6.


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Effect of LPS on renal COX-2 mRNA and protein expression in vivo. The effect of a single injection of LPS on the renal expression of COX-2 was determined in tissue samples from inner medulla and cortex. We determined COX-2 mRNA and protein 2, 4, 6, and 8 h after LPS injection by RT-PCR and Western blotting analysis, respectively. Under basal conditions, COX-2 expression was more abundant in inner medulla than in cortex, consistent with previous observations (13, 39). LPS significantly enhanced COX-2 expression at both mRNA and protein levels in the inner medulla, a change that was detectable 2 h after the treatment and lasted over the next three time points. Because the values derived from densitometric analysis of the four time points were not significantly different, the data were pooled. The average increase in COX-2 mRNA and protein in inner medulla after LPS treatment was twofold and 2.5-fold, respectively. In contrast, COX-2 expression in the cortex at either mRNA or protein levels was not significantly altered by LPS. Figure 1 and Fig. 2 show representative gels of the effect of LPS on COX-2 mRNA expression in inner medulla and COX-2 protein expression in both cortex and inner medulla, respectively.


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Fig. 1.   Phosphor image of RT-PCR amplification products of COX-2 and beta -actin mRNAs in the same RNA preparation of renal inner medulla of animals treated by lipopolysaccharide (LPS) or vehicles. PCR was run on 3 dilutions of cDNA, with 1:10, 1:100, and 1:1,000 for COX-2, and with 1:100, 1:1,000, and 1:10,000 for beta -actin.



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Fig. 2.   Western blotting analysis of COX-2 from cortex and inner medulla of Sprague-Dawley rats treated by LPS (40 mg/kg) or vehicles. Lanes for cortex were loaded with 200 µg protein and lanes for inner medulla were loaded with 100 µg protein from tissue homogenates. Visualized with phosphorimager. Representative data from total of 32 animals (control and experimental groups, 16 in each).

Effect of LPS on COX-2 promoter activity in vivo. COX-2 promoter transgenic mice were created using a 2.7-kb rat COX-2 flanking sequence fused upstream of a nuclear localization signal and lacZ. Genotyping was performed by PCR. Primers were selected to span the junction region between promoter sequence and lacZ gene, thus amplifying a product specific to the chimeric construct (Fig. 3A). The predicted 450-bp products were detected in tail DNA from the transgenic mice, but not in that of CD-1 mice (Fig. 3B). Under basal conditions, beta -galactosidase activity of both COX-2 transgenic and wild-type mice, as well as of CD-1 mice, was detectable at low levels in cortex and outer medulla, but not in inner medulla, despite a 12-h incubation. In contrast, LPS treatment induced a marked increase in the beta -galactosidase activity in the kidney, predominately in inner medulla of a transgenic founder mouse (Fig. 4, A and B). In the example shown in Fig. 4, the intense staining was detected within 15 min after the incubation with X-gal. A similar pattern of staining was also observed in three other LPS-treated F1 mice from a separate founder mouse (Fig. 4C), although intensity of staining varied.


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Fig. 3.   A: graph of construct and primer design for PCR genotyping. NLS, nuclear localization signal. B: representative agarose gel showing PCR genotyping result. Lane 1, PCR on tail DNA isolated from transgenic mice; lane 2, PCR on tail DNA isolated from CD-1 mice; lane 3, PCR on COX-2 promoter-lacZ construct.



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Fig. 4.   A: X-gal staining from kidney of COX-2 trangenic mice treated with LPS. Transgenic mice were treated with 2 doses of LPS intraperitoneally injected at 12 and 2 h before death (1 mg/kg each). Kidney slices were stained with X-gal after fixation with 4% paraformaldehyde through heart. Kidney staining from LPS-treated founder mouse is shown. B: section from same animal. C: section of renal medulla from representative LPS-treated F1 mice from a separate founder mouse.

Distribution of CD14 and LBP mRNAs in kidney regions. RT-PCR for CD14, LBP, and beta -actin was performed on 1 µg of total RNA from cortex and inner medulla of adult Sprague-Dawley rats (Fig. 5). The identity of the products was confirmed by restriction digest (data not shown). CD14 mRNA was abundantly present in inner medulla, whereas the signal was barely detectable in cortex. LBP mRNA was present in both regions, with a modestly higher abundance in inner medulla than in cortex.


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Fig. 5.   Phosphor image of RT-PCR products of CD14, LPS binding protein (LBP), and beta -actin from the same cDNA derived from cortex and inner medulla of 3 adult Sprague-Dawley rats.

Localization of CD14 in microdissected nephron segments. We determined CD14 mRNA expression in microdissected nephron segments using RT-PCR. An abundant amount of CD14 mRNA was consistently detected in inner medullary collecting ducts (IMCD; Fig. 6). Low levels of expression were also regularly detected in glomerulus. Products were not consistently observed in proximal convoluted tubule, cortical collecting ducts, cortical thick ascending limb, or arcuate artery.


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Fig. 6.   Phosphor image of RT-PCR products of CD14 and beta -actin from microdissected nephron segments. Representative of 3 experiments from 3 adult Sprague-Dawley rats. cTAL, cortical thick ascending limb; CCD, cortical collecting duct; PCT, proximal convoluted tubule; IMCD, inner medullary collecting duct.

mRNA expression in cultured cells of kidney origin. mRNA expression of CD14 was assayed in cultured cells of renal origin, including two types of cortical cells (mesangial cells and M1 cells) and two types of medullary cells (mIMCD-K2 and RMIC). Total RNA from confluent cells was isolated and was subjected to RT-PCR. The CD14 signal (from undiluted cDNA) was abundantly present in mIMCD-K2 and RMIC, the two medullary cells, but the signal was only detected at an ~100-fold lower abundance in mesangial and M1 cells, the two cortical cell lines (Fig. 7), consistent with the regional distribution pattern of CD14 shown in Fig. 5.


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Fig. 7.   mRNA expression of CD14 in cultured cells of renal origin. Confluent cells were harvested with TRI Reagent for isolation of RNA. One microgram of RNA was reverse transcribed and cDNA was subsequently precipitated with ethanol and dissolved in TE buffer (10 mM Tris and 1 mM EDTA, pH 8). PCR for CD14 and beta -actin was run on undiluted and 1:100 diluted cDNA, respectively. Shown is PCR product obtained from cDNA from 2 types of cortical cells (MS, mesangial cells; M1, cells derived from mouse cortical collecting ducts) and 2 types of medullary cells [NK2, mouse inner medullary collecting duct cells (mIMCD-K2); RMIC, renal medullary interstitial cells].

Effect of LPS on COX-2 and CD14 mRNA expression in cultured mIMCD-K2 cells. Confluent mIMCD-K2 cells were serum deprived for 12 h and then treated with LPS (100 ng/ml) for 4 and 16 h. PCR for CD14 and COX-2 was performed on 1:10 and 1:100 diluted cDNA, ,and PCR for beta -actin was performed on 1:100 and 1:1,000 diluted cDNA. Treatment of LPS (100 ng/ml) significantly stimulated both CD14 and COX-2 mRNA expression in a time-dependent manner. Stimulation of CD14 peaked at 4 h and declined at 16 h (Fig. 8), whereas stimulation of COX-2 occurred at 4 h and peaked at 16 h after the treatment (Fig. 8). To test the role of CD14 in mediation of LPS-stimulated COX-2 expression, we used CD14 antibody to neutralize CD14. As shown in Fig. 9, addition of the CD14 monoclonal antibody significantly reduced LPS-stimulated COX-2 mRNA expression in cultured mIMCD-K2 cells.


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Fig. 8.   Phosphor image of RT-PCR products of COX-2, CD14, and beta -actin from cDNA from cultured mIMCD-K2 cells treated by vehicle or LPS (100 ng/ml) for indicated period of time. Each PCR was run on 2 dilutions of cDNA, with 1:10 and 1:100 dilutions for CD14 and COX-2 and 1:100 and 1:1,000 dilutions for beta -actin.



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Fig. 9.   Effect of addition of CD14 monoclonal antibody on LPS-stimulated COX-2 mRNA expression in cultured mIMCD-K2 cells. Cells were untreated or treated with LPS (100 ng/ml) or with LPS + CD14 monoclonal antibody (50 ng/ml) for 12 h. One microgram of total RNA was subjected to RT-PCR for COX-2 and beta -actin.


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In contrast to the constitutive presence of COX-1 in many organs and tissues, COX-2 is a cyclooxygenase isozyme that is characterized by absent or low expression under resting conditions and whose production is dependent on stimulation by cytokines, mitogens, and a variety of tissue-specific activators (7). However, COX-2 has been found in the kidney in the absence of specific interventions that typically stimulate its expression. The vast majority of renal constitutive COX-2 production in rats and humans is found in the medulla, both in RMIC and collecting duct cells (39). In addition, a highly localized expression of COX-2 in macula densa cells and/or surrounding thick ascending limb cells is also present in the renal cortex (39). Commensurate with the character of COX-2 as a highly inducible enzyme, renal expression of COX-2 mRNA, as well as COX-2 protein, was found to be regulated by a number of interventions, including changes in dietary salt intake and ureteral obstruction (30, 39). The current study was designed to determine the effects of in vivo and in vitro LPS treatment on renal COX-2 expression because LPS treatment has been shown to be a potent stimulator of de novo expression of COX-2 in cells that do not express it under resting conditions (17, 22).

Our data show that LPS caused a sustained upregulation of COX-2 mRNA and protein in the renal medulla, but not in the cortex. The inner medullary cells affected by LPS include IMCD cells, based on the present RT-PCR determinations in cultured mIMCD-K2 cells. The time course of LPS-stimulated COX-2 expression is similar to that reported earlier for induction of the enzyme by LPS in macrophages (38). Upregulation of COX-2 activity is consistent with several reports showing increased renal prostaglandin production after LPS treatment (19). In one previous study, LPS administered in a comparable dose to conscious rats did not affect the expression of COX-2 mRNA in kidney tissue (36). The present results would suggest that the stimulatory effect of LPS on medullary COX-2 may have been compensated by an opposite effect in the cortex, resulting in an absent net effect for the whole kidney.

The differential response is also seen in COX-2 promoter transgenic mice. The transgenic mice were created using a 2.7-kb rat COX-2 flanking sequence fused upstream of nuclear localization signal and lacZ. Under basal conditions, there was barely detectable beta -galactosidase activity in the kidney of the transgenic mice, not consistent with the constitutive COX-2 expression in the kidney of wild-type mice. It is possible that the regulatory elements responsible for the constitutive expression of COX-2 in the kidney are located outside the 2.7-kb flanking region of the COX-2 gene, that the transgene is not integrated correctly into the chromosome, or that the staining method is not sensitive enough to detect the low level of constitutive reporter activity. We found that these animals exhibited high responsiveness to LPS treatment by expressing the reporter gene predominately in the renal medulla, consistent with the LPS-induced COX-2 expression pattern in the kidney, suggesting that the 2.7-kb flanking region contains LPS-responsive elements. We observed a certain degree of variability in intensity of reporter activity between different founder lines that may be due to differences in integration sites in chromosomes.

The mechanisms responsible for the differential effect of LPS on COX-2 expression are unclear. However, expression of a number of genes, including COX-2 and bNOS, has been shown to be differentially regulated in the two regions; for example, salt restriction stimulates the expression of bNOS and COX-2 in the cortex, whereas salt loading stimulates their expression in renal medulla. Thus it is not unprecedented that cortical and medullary COX-2 are subject to differential regulation by LPS. However, the observation of renal expression of CD14 in the present study may provide a clue to understanding the mechanism for the differential regulation of renal COX-2 by LPS. CD14 is a glycosyl-phosphatidylinositol-anchored membrane protein that is regarded as a marker of monocyte/macrophage lineage. The expression of CD14 has been reported to be restricted to monocytes, neutrophils, and macrophages. Thus our finding of constitutive expression of CD14 in the kidney was unexpected. Consistent with predominant CD14 expression in the renal medulla, we found that CD14 expression was much more abundant in mIMCD-K2 cells and RMIC compared with cells derived from cortex, such as mesangial cells and M1 cells. These findings suggest that CD14-expressing medullary cells may behave like macrophages, being capable of responding to inflammatory stimuli with the induction of COX-2 and possibly of cytokines. In support of this idea, we demonstrated that, in the mIMCD-K2 cell line, LPS treatment significantly induced COX-2 mRNA expression with concomitant induction of CD14 expression. Furthermore, we significantly reduced the LPS-stimulated COX-2 expression by adding a CD14 monoclonal antibody to the culture medium. Taken together, this demonstrates the presence of both constitutive and inducible expression of CD14 in the renal medulla that is, at least in part, responsible for the site-specific induction of COX-2 in renal medulla by LPS.

Involvement of a medullary NO system may be another reason for the different response of COX-2 to LPS in cortex and medulla. The renal medulla is known to be a rich source of NO production (40), in agreement with the abundant expression of nitric oxide synthases (NOS), including the cytokine-stimulatable or inducible form of NOS, iNOS or NOS II (24). Stimulation of iNOS production by LPS may therefore be more pronounced in the medulla than in the cortex. Because NO has been shown, in a number of preparations, to stimulate COX activity (8, 32), it is conceivable that the medullary localization of COX-2 activation by LPS reflects the local interaction with inner medullary iNOS.

Mesangial cells are known to play a role in the pathogenesis of various types of glomerular nephritis. In support of this notion, mesangial cells in vitro are capable of responding to different inflammatory interleukins by producing prostaglandins. Studies by Guan et al. (14, 15) have shown that interleukin-1beta induces COX-2 expression in cultured mesangial cells through a MAPK-mediated pathway. The low expression of CD14 in mesangial cells shown in the present study suggests that COX-2 stimulation during inflammation in these cells may primarily reflect an indirect response to cytokines such as interleukins rather than a direct response to LPS. It was an unexpected finding, however, that LPS did not detectably increase mRNA and protein levels of COX-2 in the renal cortex, suggesting a complex mechanism for the in vivo regulation of COX-2. It is possible that COX-2 in mesangial cells in vivo is stimulated by LPS through an indirect mechanism. The stimulation of COX-2 in mesangial cells may be compensated by inhibition of expression in tubular segments. In preliminary observations, we found that COX-2 expression in thick ascending limb was downregulated by LPS (data not shown). Future studies need to examine the detailed mechanism for the differential response at the cellular level.

The kidney is an organ that serves excretory and endocrine functions, but it has been recognized as an organ important in immune responsiveness as well. In certain aspects, differentiated kidney cells resemble immune cells with a potential to produce different cytokines in response to proinflammatory stimuli. For example, isolated renal interstitial cells in culture have been shown to produce cytokines, whereas skin fibroblast cells in the same condition did not (21). Clinical studies have shown that renal failure is frequently accompanied by severe infections that, in many instances, cannot be reversed by dialysis. Loss of renal immune function is likely to contribute to the infections seen in renal failure. On the other hand, disturbance of the renal immune system is known to be linked to various types of acute or chronic nephritis. Demonstration of CD14 in renal medulla and its role in LPS-stimulated medullary COX-2 expression is strong evidence in support of an important role of the kidney, particularly of the renal medulla, in immune response.

In summary, these studies report differential regulation of COX-2 expression in renal cortex and inner medulla by LPS. LPS stimulates COX-2 expression only in the renal inner medulla and not in the cortex. CD14, the LPS receptor, was found constitutively and selectively expressed in the inner medulla and in cultured cells derived from renal medulla. In the mIMCD-K2 cell line, LPS significantly induced COX-2 mRNA expression, with concomitant induction of CD14 and the LPS-stimulated COX-2 expression was reduced by the addition of CD14 monoclonal antibody to the culture medium. These results demonstrate that LPS selectively stimulates COX-2 expression in renal inner medulla through a CD14-dependent mechanism.


    ACKNOWLEDGEMENTS

We gratefully acknowledge Bruce Stanton for providing mIMCD-K2 cells and Richard Palmiter for providing pnlacf plasmid.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-37448, DK-39255, and DK-40042. Support was provided, in part, by the General Clinical Research Center (GCRC) at the University of Michigan, funded by a grant (M01RR00042) from the National Center for Research Resources, National Institutes of Health, US Public Health Service.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: J. Schnermann, NIDDK, NIH, Bldg. 10, Rm. 4DA51, 10 Center Dr., Bethesda, MD 20892-1370 (E-mail: jurgens{at}intra.niddk.nih.gov).

Received 27 August 1998; accepted in final form 3 March 1999.


    REFERENCES
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REFERENCES

1.   Anderson, G. D., S. D. Hauser, K. L. McGarity, M. E. Bremer, P. C. Isakson, and S. A. Gregory. Selective inhibition of cyclooxygenase (COX)-2 reverses inflammation and expression of COX-2 and interleukin 6 in rat adjuvant arthritis. J. Clin. Invest. 97: 2672-2679, 1996[Abstract/Free Full Text].

2.   Badr, K. F. Sepsis-associated renal vasoconstriction: potential targets for future therapy. Am. J. Kidney Dis. 20: 207-213, 1992[Medline].

3.   Breyer, M. D., and Y. Ando. Hormonal signaling and regulation of salt and water transport in the collecting duct. Annu. Rev. Physiol. 56: 711-739, 1994[Medline].

4.   Chao, H. S., A. M. Poisner, R. Poisner, and S. Handwerger. Lipopolysaccharides inhibit prolactin and renin release from human decidual cells. Biol. Reprod. 50: 210-214, 1994[Abstract].

5.   Chou, S. Y., J. G. Porush, and P. F. Faubert. Renal medullary circulation: hormonal control. Kidney Int. 37: 1-13, 1990[Medline].

6.   Dentener, M. A., V. Bazil, E. J. Von Asmuth, M. Ceska, and W. A. Buurman. Involvement of CD14 in lipopolysaccharide-induced tumor necrosis factor-alpha , IL-6 and IL-8 release by human monocytes and alveolar macrophages. J. Immunol. 150: 2885-2891, 1993[Abstract/Free Full Text].

7.   DeWitt, D., and W. L. Smith. Primary structure of prostaglandin G/H synthase from sheep vesicular gland determined from the complementary DNA sequence. Proc. Natl. Acad. Sci. USA 85: 1412-1416, 1988[Abstract].

8.   Di Rosa, M., A. Ialenti, A. Ianaro, and L. Sautebin. Interaction between nitric oxide and cyclooxygenase pathways. Prostaglandins Leukot. Essent. Fatty Acids 54: 229-238, 1996[Medline].

9.   Feng, L., W. Sun, Y. Xia, W. W. Tang, P. Chanmugam, E. Soyoola, C. B. Wilson, and D. Hwang. Cloning of two isoforms of rat cyclooxygenase: differential regulation and their expression. Arch. Biochem. Biophys. 307: 361-368, 1993[Medline].

10.   Ferrero, E., D. Jiao, B. Z. Tsuberi, L. Tesio, G. W. Rong, A. Haziot, and S. M. Goyert. Transgenic mice expressing human CD14 are hypersensitive to lipopolysaccharide. Proc. Natl. Acad. Sci. USA 90: 2380-2384, 1993[Abstract].

11.   Furst, D. E. Meloxicam: selective COX-2 inhibition in clinical practice. Semin. Arthritis Rheum. 26: 21-27, 1997[Medline].

12.   Galea, E., D. J. Reis, E. S. Fox, H. Xu, and D. L. Feinstein. CD14 mediate endotoxin induction of nitric oxide synthase in cultured brain glial cells. J. Neuroimmunol. 64: 19-28, 1996[Medline].

13.   Guan, Y., M. Chang, W. Cho, Y. Zhang, R. Redha, L. Davis, S. Chang, R. N. DuBois, C. M. Hao, and M. Breyer. Cloning, expression, and regulation of rabbit cyclooxygenase-2 in renal medullary interstitial cells. Am. J. Physiol. 273 (Renal Physiol. 42): F18-F26, 1997[Abstract/Free Full Text].

14.   Guan, Z., S. Y. Buckman, L. D. Baier, and A. R. Morrison. IGF-I and insulin amplify IL-1beta -induced nitric oxide and prostaglandin biosynthesis. Am. J. Physiol. 274 (Renal Physiol. 43): F673-F679, 1998[Abstract/Free Full Text].

15.   Guan, Z., S. Y. Buckman, A. P. Pentland, D. J. Templeton, and A. R. Morrison. Induction of cyclooxygenase-2 by the activated MEKK1 -> SEK1/MKK4 -> p38 mitogen-activated protein kinase pathway. J. Biol. Chem. 273: 12901-12908, 1998[Abstract/Free Full Text].

16.   Hershman, H. R. Prostaglandin synthase 2. Biochim. Biophys. Acta 1299: 125-140, 1996[Medline].

17.   Hwang, D., B. C. Jang, G. Yu, and M. Boudreau. Expression of mitogen-inducible cyclooxygenase induced by lipopolysaccharide: mediation through both mitogen-activated protein kinase and NF-kappa B signaling pathways in macrophages. Biochem. Pharmacol. 54: 87-96, 1997[Medline].

18.   Johnson, J. P., and M. D. Rokaw. Sepsis or ischemia in experimental acute renal failure: what have we learned? New Horiz. 3: 608-614, 1995[Medline].

19.   Jonasson, H., S. Basu, B. Andersson, and H. Kindahl. Renal excretion of prostaglandin metabolites, arginine vasopressin, and sodium during endotoxin and endogenous pyrogen induced fever in the goat. Acta Physiol. Scand. 120: 529-536, 1984[Medline].

20.   Kendall, S. K., D. F. Gordon, T. S. Birkmeier, D. Petrey, V. D. Sarapura, K. S. O'Shea, W. M. Wood, R. V. Lloyd, E. C. Ridgway, and S. A. Camper. Enhancer-mediated high-level expression of mouse pituitary glycoprotein hormone alpha -subunit transgene in thyrotropes, gonadotropes, and developing pituitary gland. Mol. Endocrinol. 8: 1420-1433, 1994[Abstract].

21.   Lonnemann, G., G. Engler-Blum, G. A. Muller, K. M. Koch, and C. A. Dinarello. Cytokines in human renal interstitial fibrosis. II. Intrinsic interleukin (IL)-1 synthesis and IL-1-dependent production of IL-6 and IL-8 by cultured kidney fibroblasts. Kidney Int. 47: 845-854, 1995[Medline].

22.   Maloney, C. G., W. A. Kutchera, K. H. Albertine, T. M. McIntyre, S. M. Prescott, and G. A. Zimmerman. Inflammatory agonists induce cyclooxygenase type 2 expression by human neutrophils. J. Immunol. 160: 1402-1410, 1998[Abstract/Free Full Text].

23.   Matteson, E. L. Recent clinical trials in the rheumatic diseases. Curr. Opin. Rheumatol. 9: 95-101, 1997[Medline].

24.   Mattson, D. L., and D. J. Higgins. Influence of dietary sodium intake on renal medullary nitric oxide synthase. Hypertension 27: 688-692, 1996[Abstract/Free Full Text].

25.   Merlie, J. P., D. Fagan, J. Mudd, and P. Needleman. Isolation and characterization of the complementary DNA for sheep seminal vesicle prostaglandin endoperoxide synthase (cyclooxygenase). J. Biol. Chem. 263: 3550-3553, 1988[Abstract/Free Full Text].

26.   Noble, S., and J. A. Balfour. Meloxicam. Drugs 51: 424-430, 1996[Medline].

27.   O'Banion, M. K., V. D. Winn, and D. A. Young. cDNA cloning and functional activity of a glucocorticoid-regulated inflammatory cyclooxygenase. Proc. Natl. Acad. Sci. USA 89: 4888-4892, 1992[Abstract].

28.   Ohtani, R., Y. Ohashi, K. Muranaga, N. Itoh, and H. Okamoto. Changes in activity of the renin-angiotensin system of the rat by induction of acute inflammation. Life Sci. 44: 237-241, 1989[Medline].

29.   Ozsan, K., V. Icoz, and R. K. Turker. Release of labile cyclo-oxygenase products of arachidonic acid from kidney by endotoxin. Experientia 40: 815-816, 1984[Medline].

30.   Park, J. M., T. Yang, L. J. Arend, A. M. Smart, J. B. Schnermann, and J. P. Briggs. Cyclooxygenase-2 is expressed in bladder during fetal development and stimulated by outlet obstruction. Am. J. Physiol. 273 (Renal Physiol. 42): F538-F544, 1997[Medline].

31.   Rietschel, E. T., J. Schletter, B. Weidemann, V. El-Samalouti, T. Mattern, U. Zahringer, U. Seydel, H. Brade, H. D. Flad, S. Kusumoto, D. Gupta, R. Dziarski, and A. J. Ulmer. Lipopolysaccharide and peptidoglycan: CD14-dependent bacterial inducers of inflammation. Microb. Drug Resist. 4: 37-44, 1998.[Medline]

32.   Salvemini, D. Regulation of cyclooxygenase enzymes by nitric oxide. Cell. Mol. Life Sci. 53: 576-582, 1997[Medline].

33.   Schnermann, J. Juxtaglomerular cell complex in the regulation of renal salt excretion. Am. J. Physiol. 274 (Regulatory Integrative Comp. Physiol. 43): R263-R279, 1998[Abstract/Free Full Text].

34.   Stein, J. H. Regulation of the renal circulation. Kidney Int. 38: 571-576, 1990[Medline].

35.   Sun, D., N. Nguyen, T. R. DeGrado, M. Schwaiger, and F. C. Brosius III. Ischemia induces translocation of the insulin-responsive glucose transporter GLUT4 to the plasma membrane of cardiac myocytes. Circulation 89: 793-798, 1994[Abstract].

36.   Takahashi, S., N. Futaki, M. Yokoyama, Y. Yamakawa, I. Arai, S. Higuchi, and S. Otomo. Expression of prostaglandin H synthase-2 in endotoxic shock induced in rats. Arch. Int. Pharmacodyn. Ther. 330: 102-115, 1995[Medline].

37.   Ulevitch, R. J., and P. S. Tobias. Receptor-dependent mechanisms of cell stimulation by bacterial endotoxin. Annu. Rev. Immunol. 13: 437-457, 1995[Medline].

38.   Wilborn, J., D. L. DeWitt, and M. Peters-Golden. Expression and role of cyclooxygenase isoforms in alveolar and peritoneal macrophages. Am. J. Physiol. 268 (Lung Cell. Mol. Physiol. 12): L294-L301, 1995[Abstract/Free Full Text].

39.   Yang, T., I. Singh, H. Pham, D. Sun, A. Smart, J. B. Schnermann, and J. P. Briggs. Regulation of cyclooxygenase expression in the kidney by dietary salt. Am. J. Physiol. 274 (Renal Physiol. 43): F481-F489, 1998[Abstract/Free Full Text].

40.   Zhou, A. P., and A. W. Cowley, Jr. Nitric oxide in the renal cortex and medulla. Hypertension 29: 194-198, 1997[Abstract/Free Full Text].


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