Statin stimulates bone morphogenetic protein-2, aggrecan,and type 2 collagen gene expression and proteoglycan synthesis in rat chondrocytes

Statins increase bone morphogenetic protein-2 (BMP-2) mRNA expression and subsequently increase new bone formation in vitro. However, the action of statins on the BMP-2 mRNA regulation of cartilage matrix synthesis by chondrocytes is unknown. We evaluated regulation of BMP-2, aggrecan, and type II collagen (COL2) mRNA and ³⁵S-labeled proteoglycan (PG) synthesis by mevastatin using cultured chondrocytes obtained from articular cartilage of fetal rats. Expression of BMP-2, aggrecan, and COL2 mRNAs were increased in the presence of 2µM mevastatin on day 2. However, longer (10 day) culture in the presence of the drug decreased the expression of these mRNAs. PG synthesis was increased 3 days after treating the cells with mevastatin, which was also decreased with longer (10 day) mevastatin treatment. These results suggest that mevastatin increases mRNA expression of BMP-2, aggrecan, and COL2 as well as PG synthesis by fetal rat chondrocytes early in the treatment period. We suggest that statins have implications for fracture and cartilage repair.     

Calcium signaling is required for ultrasound-stimulated aggrecan synthesis by rat chondrocytes.

Low-intensity ultrasound accelerates fracture healing in humans. In rat femur fracture models, ultrasound advanced healing is associated with increased proteoglycan expression. Here we report that ultrasound stimulation of primary rat chondrocytes elevated the intracellular concentration of calcium [Ca2+]i. The [Ca2+]i increase was rapid and transient at lower pressures (175-320 kPa), but rapid and sustained at higher ultrasound exposures (350-500 kPa). Chelating internal [Ca2+]i with 1,2-bis(2-aminophenoxy) ethane-N-N-N',N'-tetraacetic acid (BAPTA-AM), stopping the Ca2+/ATP-ase induced mitochondrial release of [Ca2+]i with Thapsigargin, or removing [Ca2+]i from the medium with EGTA inhibited the stimulatory effects of ultrasound on proteoglycan synthesis. These results imply that ultrasound-stimulated synthesis of cell matrix proteoglycan, associated with accelerated fracture healing, is mediated by intracellular calcium signaling.     

Low-intensity ultrasound stimulates endochondral ossification in vitro.

Animal and clinical studies have shown an acceleration of bone healing by the application of low-intensity ultrasound. The objective of this study was to examine in vitro the influence of low-intensity ultrasound on endochondral ossification of 17-day-old fetal mouse metatarsal rudiments. Forty-six triplets of paired metatarsal rudiments were resected 'en block' and cultured for 7 days with and without low-intensity ultrasound stimulation (30 mw/cm2). At days 1, 3, 5, and 7, the total length of the metatarsal rudiments, as well as the length of the calcified diaphysis were measured. Histology of the tissue was performed to examine its vitality. The increase in length of the calcified diaphysis during 7 days of culture was significantly higher in the ultrasound-treated rudiments compared to the untreated controls (P = 0.006). The growth of the control diaphysis was 180 +/- 30 microm (mean +/- SEM), while the growth of the ultrasound-treated diaphysis was 530 +/- 120 microm. The total length of the metatarsal rudiments was not affected by ultrasound treatment. Histology revealed a healthy condition of both ultrasound-treated and control rudiments. In conclusion, low-intensity ultrasound treatment stimulated endochondral ossification of fetal mouse metatarsal rudiments. This might be due to stimulation of activity and/or differentiation of osteoblasts and hypertrophic chondrocytes. Our results support the hypothesis that low-intensity ultrasound activates ossification via a direct effect on osteoblasts and ossifying cartilage.     

Exposure to low-intensity ultrasound increases aggrecan gene expression in a rat femur fracture model

The effects of ultrasound stimulation on various parameters of bone repair after diaphyseal injury were assessed in a standard rat femur fracture model. Bilateral closed femoral fractures were made in 79 skeletally mature male Long-Evans rats. An ultrasound signal consisting of a 200 microsecond burst sine wave of 0.5 MHz repeating at 1 kHz, with an intensity of 50 or 100 mW/cm² spatial and temporal average, was applied to one fracture in each animal. The contralateral fracture was not exposed to ultrasound and served as a control. Mechanical testing of the healing fracture was performed 3 weeks after injury. In fractures treated with a 50 mW/cm² ultrasound signal, the average maximum torque (223.5 ± 50.5 Nmm compared with 172.6 ± 54.9 Nmm, p = 0.022, paired t test) and average torsional stiffness (13.0 ± 3.4 Nmm/° compared with 9.5 ± 2.9 Nmm/°, p = 0.017) were significantly greater in treated than in control fractures. In animals treated with a 100 mW/cm² ultrasound signal, the average maximum torque and torsional stiffness were greater in treated than in control fractures, but this trend did not reach statistical significance. Biochemical analysis of callus in ultrasound-treated and control fractures failed to demonstrate significant differences in cell number, collagen content, or calcium content. Evaluation of gene expression in fractures treated with 50 mW/cm² ultrasound demonstrated a shift in the expression of genes associated with cartilage formation; aggrecan gene expression was significantly higher on day 7 after fracture and significantly lower on day 21 (p = 0.033 and 0.035, respectively). αl (11) procollagen gene expression was similarly modified, but this trend did not reach statistical significance. Expression of genes coding for bone-related proteins, including α1(I) procollagen, bone γ-carboxyglutamic acid protein, alkaline phosphatase, and transforming growth factor-β1, did not differ between ultrasound-treated and control fractures. These data suggest that ultrasound stimulation increased the mechanical properties of the healing fracture callus by stimulating earlier synthesis of extracellular matrix proteins in cartilage, possibly altering chondrocyte maturation and endochondral bone formation.     

Time-dependent aggrecan gene expression of articular chondrocytes in response to hyperosmotic loading

OBJECTIVE: To investigate the effects of increasing extracellular osmolality on aggrecan gene expression and cell size in cultured chondrocytes. DESIGN: Aggrecan promoter activity and mRNA levels were measured in bovine monolayer chondrocytes subjected to hyperosmotic loading for different time periods, using transient transfection assays or RT-PCR. Cell size changes were also determined using an epifluorescence microscopy system. RESULTS: Hyperosmotic loading for 24 h suppressed aggrecan promoter activity and mRNA levels approximately two-fold. However no suppression of promoter activity was observed when exon 1 was deleted from the human aggrecan promoter construct. Osmotic regulation of aggrecan gene expression was time-dependent and found to correlate with cell shrinking and swelling. No suppression in promoter activity was observed when the hyperosmotic stimulus was applied in a cyclic manner, or when serum was present in the culture medium. CONCLUSION: Hyperosmotic loading regulates aggrecan gene expression and cell size in isolated chondrocytes. Osmotic regulation of gene expression is also affected by the time-varying nature of loading and the presence of serum.     

Low-density lipoprotein stimulates mesangial cell proteoglycan and hyaluronan synthesis.

BACKGROUND: Hyperlipidaemia leads to glomerulosclerosis in small mammals and may contribute to progressive renal disease in man. One prominent feature of lipid-induced glomerular injury in animal models is the accumulation of mesangial matrix. These studies were designed to investigate whether low-density lipoprotein (LDL) enhanced mesangial cell (MC) matrix deposition by modulating the production of proteoglycans (PG) and hyaluronan (HA). METHODS: Growth arrested human MC were metabolically labelled with either 50 microCi/ml Na(2)[(35)S]sulphate or 25 microCi/ml [(3)H]glucosamine and stimulated with LDL (10-100 microg/ml). The radiolabelled PG and HA extracted from the cell layer and the culture medium were isolated, quantified and characterized. Comparison of the PG core proteins synthesized by MC was carried out using Western blot analysis. RESULTS: LDL stimulation led to a dose- and time-dependent increase in [(35)S]sulphate incorporation into PG in the culture medium and to a lesser extent in the cell layer. Analysis of the glycosaminoglycan (GAG) chains showed no difference in either their size or charge. Enzyme digestion studies demonstrated that the synthesis of both chondroitin sulphate PG (CSPG) and heparan sulphate PG (HSPG) was enhanced as was the production of the core proteins of versican (a large CSPG), perlecan (a basement membrane HSPG) and to a lesser extent decorin (a small dermatan sulphate PG (DSPG)). An increase in HA synthesis was also demonstrated in [(3)H]glucosamine labelled cells following LDL stimulation. CONCLUSION: LDL selectively enhances the synthesis of specific PG and HA by mesangial cells. Such effects may contribute to the expansion of the mesangial matrix and modify cell-matrix interactions in lipid-induced renal damage.     

Low-intensity laser irradiation stimulates bone nodule formation via insulin-like growth factor-I expression in rat calvarial cells

BACKGROUND AND OBJECTIVE: We previously reported that low-intensity laser irradiation stimulated bone nodule formation through enhanced cellular proliferation and differentiation. However, the mechanisms of irradiation are unclear. Thus, we attempted to determine the responsibility of insulin-like growth factor (IGF)-I for the action observed. STUDY DESIGN/MATERIALS AND METHODS: Osteoblast-like cells were isolated from fetal rat calvariae and cultured with rat recombinant (r) IGF-I, IGF-I-antibody (Ab), and/or the cells were irradiated once (3.75 J/cm(2)) with a low-intensity Ga-Al-As laser (830 nm). The number and area of bone nodules formed in the culture were analyzed, and IGF-I expression was also examined. RESULTS: Treatment with rIGF-I significantly stimulated the number and area of bone nodules. This stimulatory effect was quite similar to those by laser irradiation, and this stimulation was abrogated dose-dependently by treatment with IGF-I-Ab. Moreover, laser irradiation significantly increased IGF-I protein and gene expression. CONCLUSION: The stimulatory effect of bone nodule formation by low-intensity laser irradiation will be at least partly mediated by IGF-I expression.     

AG-041R stimulates cartilage matrix synthesis without promoting terminal differentiation in rat articular chondrocytes

OBJECTIVE: AG-041R, a novel indolin-2-one derivative, has recently been demonstrated to induce systemic hyaline cartilage hyperplasia in rats. The aim of this study was to characterize its anabolic actions on chondrocytes. DESIGN: Chondrocytes were isolated from knee joints of 5-week-old SD rats. Effects of AG-041R on cartilage matrix synthesis were examined by measuring [(35)S]sulfate incorporation into proteoglycans, Alcian blue staining, and Northern blotting of cartilage matrix genes. ALP activity, mineral deposition and the expression of markers for hypertrophic chondrocytes, were assessed for terminal differentiation of chondrocytes. Roles of endogenous TGF-beta/BMPs and MEK1/Erk signaling in the action of AG-041R were investigated using the neutralizing soluble receptors and the MEK1 inhibitor. RESULTS: AG-041R accelerated proteoglycan synthesis assessed by both [(35)S]sulfate incorporation and Alcian blue stainable extracellular matrix accumulation. It also up-regulated the gene expression of type II collagen and aggrecan, as well as tenascin, a marker for articular cartilage. In contrast, AG-041R suppressed ALP activity, mineralization, and the gene expression of type X collagen and Cbfa1, indicating that AG-041R prevents chondrocyte terminal differentiation. AG-041R increased in BMP-2 mRNA, and the neutralizing soluble receptor for BMPs reversed the stimulatory effects of AG-041R on cartilage matrix synthesis. Moreover, AG-041R activated MEK1/Erk pathway, which was revealed to prevent chondrocyte terminal differentiation. CONCLUSION: AG-041R stimulates cartilage matrix synthesis without promoting terminal differentiation in rat articular chondrocytes, which is mediated at least in part by endogenous BMPs and Erk. The data demonstrates that AG-041R has a potential to be a useful therapeutic agent for articular cartilage disorders.     

Dexamethasone stimulates expression of C-type Natriuretic Peptide in chondrocytes

Background  

Growth of endochondral bones is regulated through the activity of cartilaginous growth plates. Disruption of the physiological patterns of chondrocyte proliferation and differentiation – such as in endocrine disorders or in many different genetic diseases (e.g. chondrodysplasias) – generally results in dwarfism and skeletal defects. For example, glucocorticoid administration in children inhibits endochondral bone growth, but the molecular targets of these hormones in chondrocytes remain largely unknown. In contrast, recent studies have shown that C-type Natriuretic Peptide (CNP) is an important anabolic regulator of cartilage growth, and loss-of-function mutations in the human CNP receptor gene cause dwarfism. We asked whether glucocorticoids could exert their activities by interfering with the expression of CNP or its downstream signaling components.     

Dexamethasone stimulates expression of C-type Natriuretic Peptide in chondrocytes

Background

Endostatin is a C-terminal fragment of collagen XVIII which is a component of basement membranes with the structural properties of both collagens and proteoglycans. Endostatin has a major role in angiogenesis which is intimately associated with bone development and remodeling. Signaling between the endothelial cells and the bone cells, for example, may have a role in recruitment of osteoclastic precursor cells. Our study aims at exploring a possibility that endostatin, either as a part of basement membrane or as a soluble molecule, may control osteoclastogenesis and osteoclastic bone resorption in vitro.

Methods

Rat pit formation assay was employed in order to examine the effect of endostatin alone or in combination with vascular endothelial growth factor-A (VEGF-A) on bone resorption in vitro. Effect of these agents on osteoclast differentiation in vitro was also tested. Osteoclastogenesis and the number of osteoclasts were followed by tartrate resistant acid phosphatase (TRACP) staining and resorption was evaluated by measuring the area of excavated pits.

Results

Endostatin inhibited the VEGF-A stimulated osteoclastic bone resorption, whereas endostatin alone had no effect on the basal resorption level in the absence of VEGF-A. In addition, endostatin could inhibit osteoclast differentiation in vitro independent of VEGF-A.

Conclusion

Our in vitro data indicate that collagen XVIII/endostatin can suppress VEGF-A induced osteoclastic bone resorption to the basal level. Osteoclastogenesis is also inhibited by endostatin. The regulatory effect of endostatin, however, is not critical since endostatin alone does not modify the basal bone resorption.     

Effects of dexamethasone on proteoglycan content and gene expression of IL-1beta-stimulated osteoarthrotic chondrocytes in vitro

We studied the effects of dexamethasone on proteoglycan (PG) concentration and gene expression in human osteoarthrotic chondrocytes stimulated with IL-1beta. Cartilage samples were taken from 7 patients with osteoarthrosis of the knee and chondrocytes cultivated in alginate beads. Dexamethasone was added in three concentrations (10(-5), 10(-6), 10(-7) M) to IL-1beta (100 pg/nL)-stimulated chondrocytes. PG concentration was estimated by a dimethylmethylene blue assay. To assess cell proliferation, DNA content was measured fluorometrically. Quantitative Lightcycler-PCR was used to estimate the mRNA levels of stromelysin-1 (MMP-3) and aggrecan (AGG). The proliferation rate was unchanged in all treatment groups. IL-1beta increased MMP-3 expression by 44% and inhibited AGG expression by 16%, but PG-concentration was reduced by 7%. The addition of dexamethasone to IL-1beta-stimulated chondrocytes further reduced the PG concentration by 19% at 10(-5) M and by 17% at 10(-7) M. The MMP-3 expression was inhibited between 27-53% and the AGG expression between 30-46% by dexamethasone. In osteoarthrotic chondrocytes, dexamethasone in an appropriate dose range reduced the expression of MMP-3 and AGG at the same time. The resulting decrease in PG concentration should be considered when using intraarticular corticosteroids to treat an osteoarthrotic joint.     

Sphingosine-1-phosphate attenuates proteoglycan aggrecan expression via production of prostaglandin E<Subscript>2</Subscript> from human articular chondrocytes

Background  

Sphingosine-1-phosphate (S1P), a downstream metabolite of ceramide, induces various bioactivities via two distinct pathways: as an intracellular second messenger or through receptor activation. The receptor for S1P (S1PR) is the family of Endothelial differentiation, sphingolipid G-protein-coupled receptor (EDG). We have here attempted to reveal the expression of EDG/S1PR in human articular chondrocytes (HAC), exploring the implications of S1P in cartilage degradation.     

The proteoglycan synthesis repertoire of rabbit chondrocytes maintained in type II collagen gels

Repair of experimental articular cartilage lesions employing cultured rabbit articular chondrocytes requires a detailed knowledge of the phenotypic stability of these cells. A suitable matrix vehicle for use in chondrocyte transplantation is a much sought-after component of any transplantation paradigm. We studied the proteoglycan synthesis repertoire of young immature rabbit articular chondrocytes maintained in chick type II collagen gels or collagen gels supplemented with recombinant human transforming growth factor-beta 1 (rhTGF beta 1). Maintenance of chondrocytes in type II collagen gels increased the percentage 35SO4-labeled proteoglycans reaching equilibrium in the A1D1 or D1 fraction of CsCl density gradient when compared to chondrocytes maintained in polystyrene microwell cultures. Although rhTGF beta 1 supplementation increased the percentage of A1D1/D1 proteoglycan by chondrocytes grown on polystyrene, rhTGF beta 1 did not augment this percentage increase in A1D1/D1 when added to collagen II gels. Rabbit chondrocytes synthesized two core proteins derived from the high-density aggregatable proteoglycans. LI and LII have apparent molecular sizes of 480 kDa and 390 kDa, respectively. Both core protein forms were found in the medium fraction, but the predominant core protein form associated with the cell fraction was LI. Maintenance of chondrocytes in collagen II gels increased synthesis of both core proteins. In addition to the large core proteins, three other core proteins with properties on SDS PAGE characteristic of the small dermatan sulfate proteoglycans, biglycan and decorin, were identified. Synthesis of these core proteins was stimulated by maintenance in collagen gels. Furthermore, they were preferentially retained in the gel matrix. Chondrocytes maintained on glass or in type II collagen gels stained with monoclonal antibodies specific for chondroitin-6-sulfate, chondroitin-4-sulfate and keratan sulfate. However, while chondrocytes grown on glass slides failed to stain with monoclonal antibody 3B3 in the absence of chondroitinase ABC digestion, chondrocytes grown in collagen II gels stained intensely in the absence of enzyme pretreatment. These results were confirmed by Western blots.     

Low-intensity pulsed ultrasound accelerates rat femoral fracture healing by acting on the various cellular reactions in the fracture callus.

Low-intensity pulsed ultrasound (LIPUS) has been shown to accelerate fracture healing in both animal models and clinical trials, but the mechanism of action remains unclear. In fracture healing, various consecutive cellular reactions occurred until repair. We investigated whether the advanced effects of LIPUS depended on the duration and timing of LIPUS treatment in a rat closed femoral fracture model to determine the target of LIPUS in the healing process. Sixty-nine Long-Evans male rats that have bilateral closed femoral fractures were used. The right femur was exposed to LIPUS (30 mW/cm2 spatial and temporal average [SATA], for 20 minutes/day), and the left femur was used as a control. Rats were divided into four groups according to timing and duration of treatment (Ph-1, days 1-8; Ph-2, days 9-16; Ph-3, days 17-24; throughout [T], days 1-24 after the fracture). Animals were killed on day 25. After radiographs and microfocus X-ray computed tomography (muCT) tomograms were taken, the hard callus area (HCA), bone mineral content (BMC) at the fracture site, and mechanical torsion properties were measured, and histological analysis was conducted. Interestingly, the maximum torque of the LIPUS-treated femur was significantly greater than that of the controls in all groups without any changes in HCA and BMC. The multiviewing of three-dimensional (3D) muCT reconstructions and histology supported our findings that the partial LIPUS treatment time was able to accelerate healing, but longer treatment was more effective. These results suggest that LIPUS acts on some cellular reactions involved in each phase of the healing process such as inflammatory reaction, angiogenesis, chondrogenesis, intramembranous ossification, endochondral ossification, and bone remodeling.     

整合素介导的软骨细胞力学信号转导与基因表达

整合素是异源二聚体组成的细胞膜黏附蛋白,它与相应的细胞外基质胶原蛋白结合是软骨细胞力学信号转导的前提.整合素一方面将细胞与细胞外基质连接在一起,维持细胞结构和力学完整性,另一方面将力学信号转化为生物化学信号并与其他细胞表面膜分子一起协调将信号传入细胞内,调控基因表达,在软骨细胞分化、生长、发育和成熟过程中发挥重要作用.整合素介导的软骨细胞力学信号转导环节的障碍或紊乱,可导致病理状态如发生骨关节炎.该文就整合素介导的软骨细胞对力学的感受,细胞内信号转导、基因表达对软骨细胞力学信号转导机制的研究作一综述.     

Induction of apoptosis of articular chondrocytes and suppression of articular cartilage proteoglycan synthesis by heat shock

We investigated cellular and matrix responses of articular cartilage to heat shock. Rat articular cartilage was pretreated at 37 degrees C for 24 h before being exposed to 48 degrees C for 10 min and subsequently incubated at 37 degrees C for 1, 2, 4, 7, 10, and 14 days. Following heat shock, a terminal deoxynucleotidyl transferase nick end labeling assay showed that articular chondrocyte apoptosis appeared at day 1, peaked at day 7, and declined by day 14. Analysis by transmission electron microscopy confirmed that the chondrocytes had characteristic morphological features of apoptosis; immunohistochemical staining revealed that caspase-3 activity in chondrocytes increased, 3-B-3-positive articular chondrocytes decreased in number, and the expression of 3-B-3 native epitope in articular chondrocytes was reduced. Safranin-O staining revealed that depletion of proteoglycans in the matrix was not found in any group. Morphological and biochemical evidence from this study suggested that heat shock at 48 degrees C induced articular chondrocyte apoptosis and suppressed proteoglycan synthesis of articular cartilage in vitro. This study thus provides evidence of the onset of osteoarthritis induced by heat shock and a basis for choosing a temperature at which malignant bone tumor cells can be killed with minimal damage to articular cartilage.     

Regulation of articular chondrocyte aggrecan and collagen gene expression by multiple growth factor gene transfer

Gene transfer is a promising approach to the delivery of chondrotrophic growth factors to promote cartilage repair. It is unlikely that a single growth factor transgene will optimally regulate these cells. The aim of this study was to identify those growth factor transgene combinations that optimally regulate aggrecan, collagen type II and collagen type I gene expression by articular chondrocytes. We delivered combinations of the transgenes encoding fibroblast growth factor-2, insulin-like growth factor I, transforming growth factor beta1, bone morphogenetic protein-2, and/or bone morphogenetic protein-7 and assessed chondrocyte responses by measuring changes in the expression of aggrecan, type II collagen and type I collagen genes. These growth factor transgenes differentially regulated the magnitude and time course of all three-matrix protein genes. In concert, the transgenes regulated matrix gene expression in an interactive fashion that ranged from synergistic to inhibitory. Maximum stimulation of aggrecan (16-fold) and type II collagen (35-fold) expression was with the combination of IGF-I, BMP-2, and BMP-7 transgenes. The results indicate that the optimal choice of growth factor genes for cell-based cartilage repair cannot be predicted from observations of individual transgenes. Rather, such gene therapy will require an empirically based selection of growth factor gene combinations.     

大鼠终板软骨细胞体外自然退变过程中Asporin基因的表达及意义

目的通过大鼠终板软骨细胞体外自然传代的退变模型,探讨大鼠终板软骨细胞体外自然退变过程中Asporin基因的表达及意义。方法取大鼠腰椎终板软骨细胞,酶消化法及自然传代法分离培养大鼠终板软骨细胞,选取P1代及P5代,体外培养6d,在倒置显微镜下观察细胞形态,利用HE染色及甲苯胺蓝染色对软骨细胞进行观察及鉴定。用RT-PCR及Westernblot检测Ⅱ型胶原、蛋白多糖及Asporin基因变化。结果随着细胞传至P5代,细胞形态上有梭形变趋势,Ⅱ型胶原（P5/P1=0.111767,P=0.009842）及蛋白多糖表达（P5/P1=0.328965,P=0.002371）明显下调,Asporin蛋白表达明显上调。结论终板软骨细胞传至P5代,细胞发生自然退变,Asporin基因表达明显上调,提示Asporin基因表达与终板软骨的退变具有重要联系。     

Subtotal nephrectomy stimulates cyclooxygenase 2 expression and prostacyclin synthesis in the rat remnant kidney

BACKGROUND: Recently, two isoforms of cyclooxygenase (COX) have been identified, a constitutive form (COX-1) and a mitogen-inducible form (COX-2). Several studies have suggested that COX is activated in renal insufficiency, but little is known about the relationship between progression of renal insufficiency and the COX isoforms. METHODS: Five-sixths-nephrectomized (NX) rats were used. 4, 8, and 12 weeks after nephrectomy, the renal cortical prostaglandin contents and the expression levels of the two isoforms of COX were determined by enzyme immunoassay and Western-blotting, respectively. The localization of COX was examined by immunohistochemistry. RESULTS: Renal cortical prostacyclin (PGI2) and COX-2 were significantly upregulated 8 and 12 weeks after NX, while COX-1 remained at the basal level. There was a high correlation between COX-2 and creatinine clearance (r = -0.845). There was also a high correlation between COX-2 and PGI2 (r = 0.816). Immunohistochemistry revealed the expression of COX-2 to be enhanced in the macula densa in NX rats. CONCLUSIONS: Renal cortical COX-2 and prostacyclin were upregulated corresponding to the progression of renal insufficiency in NX rats. These results suggest enhancement of COX-2 expression in the macula densa, perhaps stimulated by a decrease in renal blood flow which upregulates PGI2 synthesis to protect the kidney from ischemia in renal insufficiency.