Jun-Ling Wang

Angiotensin II attenuates renal cortical cyclooxygenase-2 expression

We have previously shown that in rat renal cortex, cyclooxygenase-2 (COX-2) expression is localized to cTALH cells in the region of the macula densa, and that dietary salt restriction increases COX-2 expression. Administration of the angiotensin converting inhibitor, captopril, further increased COX-2 mRNA and renal cortical COX-2 immunoreactivity, with the most pronounced expression in the macula densa. Administration of an AT1 receptor antagonist, losartan, also significantly increased cortical COX-2 mRNA expression and COX-2 immunoreactivity. Mutant mice homozygous for both Agtr1a and Agtr1b null mutations (Agtr1a-/-,Agtr1b-/-) demonstrated large increases in immunoreactive COX-2 expression inthe cTALH/macula densa. To determine whether increased COX-2expression in response to ACE inhibition mediated increases in renin production, rats were treated with captopril for one week with or without the specific COX-2 inhibitor, SC58236. Plasma renin activity increased significantly in the captropril group, and this increase was significantly inhibited by simultaneous treatment with SC58236. Thus, these studies indicated that angiotensin II inhibitors augment upregulation of renal cortical COX-2 in states of volume depletion, suggesting that negative feedback by the renin-angiotensin system modulates renal cortical COX-2 expression and that COX-2 is a mediator of increased renin production in response to inhibition of angiotension II production.

Cyclooxygenase-2-selective inhibitors impair glomerulogenesis and renal cortical development

BACKGROUND Antenatal exposure to nonsteroidal anti-inflammatory drugs (NSAIDs) has been associated with renal dysgenesis in humans. METHODS These studies characterized cyclooxygenase-2 (COX-2) versus COX-1-selective inhibition on nephrogenesis in the rodent using histomorphometry, immunohistology, and in situ hybridization. RESULTS Administration of a COX-2-selective inhibitor (SC58236), started during pregnancy until weaning, significantly impaired development of the renal cortex and reduced glomerular diameter in both mice and rats. An identical phenotype was demonstrated in COX-2 -/- mice. In contrast to its effects on the developing kidney, a COX-2 inhibitor had no effect on glomerular volume in adult mice. This effect was specific for COX-2 because maternal administration of a COX-1-selective inhibitor (SC58560) did not affect renal development despite significantly inhibiting gastric mucosal prostaglandin E2 (PGE2) synthesis in pups. The expression of COX-2 immunoreactivity peaked in the first postnatal week and was localized to S-shaped bodies and the macula densa in the cortex. Treatment with a COX-2 inhibitor during this period (from postnatal day 0 to day 21) severely reduced glomerular diameter, whereas treatment limited to pregnancy did not affect glomerular size. CONCLUSION These data demonstrate an important role for COX-2 activity in nephrogenesis in the rodent, and define a specific time period of susceptibility to these effects.

Cyclooxygenase-2 Inhibition Decreases Renin Content and Lowers Blood Pressure in a Model of Renovascular Hypertension

It has been proposed that the macula densa participates in the regulation of increased renin expression in renovascular hypertension (RVH) and that prostaglandins may be among the mediators of macula densa function. We have previously shown that in renal cortex, cyclooxygenase-2 (COX-2) expression is localized to the macula densa and surrounding cortical thick ascending limb and increases in high-renin states, such as salt restriction and angiotensin-converting enzyme inhibition. In the present studies, we examined the effect of the selective COX-2 inhibitor SC58236 on plasma renin activity (PRA) and renal renin expression in RVH in rats. The aorta was coarcted between right and left renal arteries, and animals received either SC58236 or vehicle for 1 week. At day 8, vehicle-treated coarcted rats were hypertensive (mean carotid arterial blood pressure: 138+/-3 versus 87+/-2 mm Hg in sham-operated controls; n=9 to 11; P<0.001) and exhibited a disparity of kidney size (ratio left/right kidney: 0.78+/-0.04 versus 1.02+/-0.02; n=9 to 10; P<0.001). PRA increased significantly (84.6+/-6.5 versus 9.0+/-1.4 ng angiotensin I [Ang I] per milliliter per hour; n=8 to 9; P<0.01). In the coarcted rats, neither renin mRNA expression nor renin activity of the right kidney was altered (renin/GAPDH mRNA: 1.12+/-0.05-fold levels in control rats; n=6; P=NS; renin activity: 23.4+/-1.8 versus 27.1+/-3.4 ng Ang I per hour per milligram protein; n=8 to 9; P=NS). However, the renin mRNA of the left kidney increased to 3.0+/-0.6-fold of control (n=6), and the renin activity increased to 189.0+/-28.6 ng Ang I per hour per milligram protein (n=8; P<0.01). Expression of COX-2 mRNA and immunoreactive protein increased in the affected left kidney but was not different from control in the unaffected right kidney. SC58236 treatment to coarcted rats did not affect kidney size (ratio left/right kidney: 0.79+/-0.06; n=9). However, PRA was significantly decreased compared with the vehicle-treated coarcted rats (19.8+/-2. 8 ng Ang I per milliliter per hour; n=9; P<0.01). The left kidney renin mRNA and renin content were also decreased (1.7+/-0.3-fold control; n=6; P<0.05; and 45.7+/-7.6 ng Ang I per hour per milligram protein; n=9; P<0.01, respectively), while renin mRNA and renin content of the right kidney were not altered. SC58236 lowered mean arterial blood pressure (122+/-5 mm Hg; n=14; P<0.05 compared with vehicle). A significant correlation was observed between PRA and mean blood pressure (r=0.75; P<0.01). In summary, these studies indicate that the selective COX-2 inhibitor SC58236 decreases renin production and release in RVH and suggest an important role for COX-2 regulation of the renin-angiotensin system.

Role of p38 in the regulation of renal cortical cyclooxygenase-2 expression by extracellular chloride

We have previously shown that in renal cortex, COX-2 expression is localized to macula densa and surrounding cortical thick ascending limb of Henle (cTALH). Dietary salt restriction increases local expression of COX-2, which mediates renin production and secretion. Given that decreased luminal chloride [Cl(-)] at the level of the macula densa increases renin production and secretion, we investigated the role of extracellular ion concentration on COX-2 expression. Quiescent rabbit cTALH cells were incubated in a physiological salt solution containing high or low levels of NaCl. Immunoreactive COX-2 expression increased significantly in the low NaCl solution. COX-2 expression also increased after administration of the Na(+)/K(+)/2Cl(-) cotransport inhibitor, bumetanide. Selective substitution of chloride led to increased COX-2 expression, whereas selective substitution of sodium had no effect. The p38 MAP kinase inhibitor PD169316 decreased low NaCl-induced COX-2 expression. Low-salt or low-chloride medium induced cultured cTALH to accumulate >/= 3-fold higher levels of pp38, the activated (phosphorylated) form of p38; low-salt medium also increased pJNK and pERK levels. Feeding rats a low-salt diet for 14 days induced a significant increase in renal cortical pp38 expression, predominantly in the macula densa and cTALH. These results suggest that reduced extracellular chloride leads to increased COX-2 expression, which may be mediated by activation of a p38-dependent signaling pathway.

Cyclooxygenase-2 in rat nephron development

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

Selective increase of cyclooxygenase-2 expression in a model of renal ablation.

Previous studies have suggested a possible role for prostaglandins (PGs) in mediating alterations in nephron structure and function ensuing after renal ablation. Two isoforms of cyclooxygenase (COX) have been described: constitutive (COX-1) and inducible (COX-2). We examined expression of these isoforms following subtotal renal ablation (5/6 ablation, RA) in rats. In renal cortex, COX-2 mRNA and immunoreactive protein (IP) increased progressively compared with sham-operated littermates. In contrast, there were no significant changes in COX-1 mRNA expression. In normal kidney, cortical COX-1 IP was immunolocalized predominantly to mesangial cells and collecting tubules, whereas COX-2 IP was found in a subset of cortical thick ascending limb of Henles loop (CTAL) cells in the region of the macula densa (MD). Following RA, significantly increased COX-2 IP was detected in the MD and surrounding CTAL cells. In addition, fainter immunoreactive COX-2 was detected in scattered visceral epithelial cells and mesangial cells of the glomerulus. Immunoblotting of isolated glomeruli demonstrated a selective increase of glomerular immunoreactive COX-2 expression following RA. No change of COX-1 expression was seen. To determine COX activity, isolated glomeruli were incubated with arachidonic acid and PGE2 measured by enzyme immunoassay (EIA). Compared with sham, glomeruli from 2 wk RA produced significantly more PGs. SC-58560, a selective COX-1 inhibitor, did not inhibit PG production in the remnant glomeruli at concentrations up to 10(-4) M, whereas SC-58236, a relatively selective COX-2 inhibitor, significantly inhibited PG production by RA glomeruli. In preliminary studies, to define mechanisms of altered expression of glomerular COX-2, rat mesangial cells were incubated with serum from sham or 2 wk RA. There were significant increases in COX-2 expression in response to 2 wk RA serum. In summary, these results indicate selective increases in renal cortical COX-2 expression following renal ablation.Previous studies have suggested a possible role for prostaglandins (PGs) in mediating alterations in nephron structure and function ensuing after renal ablation. Two isoforms of cyclooxygenase (COX) have been described: constitutive (COX-1) and inducible (COX-2). We examined expression of these isoforms following subtotal renal ablation (5/6 ablation, RA) in rats. In renal cortex, COX-2 mRNA and immunoreactive protein (IP) increased progressively compared with sham-operated littermates. In contrast, there were no significant changes in COX-1 mRNA expression. In normal kidney, cortical COX-1 IP was immunolocalized predominantly to mesangial cells and collecting tubules, whereas COX-2 IP was found in a subset of cortical thick ascending limb of Henles loop (CTAL) cells in the region of the macula densa (MD). Following RA, significantly increased COX-2 IP was detected in the MD and surrounding CTAL cells. In addition, fainter immunoreactive COX-2 was detected in scattered visceral epithelial cells and mesangial cells of the glomerulus. Immunoblotting of isolated glomeruli demonstrated a selective increase of glomerular immunoreactive COX-2 expression following RA. No change of COX-1 expression was seen. To determine COX activity, isolated glomeruli were incubated with arachidonic acid and PGE2 measured by enzyme immunoassay (EIA). Compared with sham, glomeruli from 2 wk RA produced significantly more PGs. SC-58560, a selective COX-1 inhibitor, did not inhibit PG production in the remnant glomeruli at concentrations up to 10-4 M, whereas SC-58236, a relatively selective COX-2 inhibitor, significantly inhibited PG production by RA glomeruli. In preliminary studies, to define mechanisms of altered expression of glomerular COX-2, rat mesangial cells were incubated with serum from sham or 2 wk RA. There were significant increases in COX-2 expression in response to 2 wk RA serum. In summary, these results indicate selective increases in renal cortical COX-2 expression following renal ablation.

Constitutive expression of cyclooxygenase-2 in rat vas deferens.

Prostaglandins, lipoid substances discovered in human semen as modulators of uterine muscle contractility, are known to play significant roles in virtually all mammalian organ systems, but their male reproductive functions are unclear. Cyclooxygenase, the rate-limiting enzyme in prostaglandin synthesis, occurs in two isoforms distinguished on the basis of constitutive (COX-1) or inducible (COX-2) expression patterns in mammalian tissues. However, in the adult rat male reproductive system, immunohistochemistry and Western and Northern analysis showed that COX-2 is the predominant isoform and is heavily localized to the epithelium of the distal vas deferens, where constitutive expression is manyfold greater than in any other organs of the body. COX-2 is not detected in the proximal one-half of the vas nor in the testis, epididymis, seminal vesicles, or prostate. Elimination of luminal sperm by vasectomy does not affect COX-2 levels, whereas castration severely depletes COX-2 and androgen replacement after castration restores COX-2, indicating that COX-2 expression in the vas is androgen dependent. Because the distal vas also comprises an extensive submucosal venous plexus connected to the penile corpora cavernosa, prostaglandins from the vas may play a role in erection.

Young SHR express increased type 1 angiotensin II receptors in renal proximal tubule

A potential role for the renin-angiotensin system (RAS) in the development and/or maintenance of hypertension in the genetic model of rat hypertension, spontaneously hypertensive rats (SHR), has been suggested by studies indicating that treatment of immature animals with angiotensin-converting enzyme (ACE) inhibitors prevents subsequent development of hypertension. Because young SHR also demonstrate RAS-dependent increased sodium retention, we examined proximal tubule type 1 angiotensin II receptor (AT1R) mRNA expression in young (4 wk) or adult (14 wk) SHR compared with age-matched Wistar-Kyoto (WKY) rats. Proximal tubules were isolated by Percoll gradient centrifugation, and AT1R mRNA expression was measured by quantitative reverse transcription-polymerase chain reaction (RT-PCR). At 14 wk, when SHR had established hypertension [mean arterial blood pressure (MAP) of SHR vs. WKY: 145 +/- 6 vs. 85 +/- 5 mmHg, n = 14-15], there were no differences in proximal tubule AT1R mRNA levels [SHR vs. WKY: 79 +/- 14 vs. 72 +/- 14 counts/min (cpm) per cpm mutant AT1R per cpm beta-actin x 10(-6), n = 6; not significant (NS)]. In contrast, in 4 wk SHR, at a time of minimal elevations in blood pressure (MAP: 70 +/- 8 vs. 63 +/- 3), SHR proximal tubule AT1R mRNA levels were 263 +/- 30% that of WKY (143 +/- 18 vs. 60 +/- 11 cpm per cpm of mutant AT1R per cpm beta-actin x 10(-6), n = 8; P < 0.005). We have recently shown that chronic ACE inhibition decreases proximal tubule AT1R expression and have also shown that chronic L-3,4-dihydroxyphenylalamine (L-DOPA) administration inhibits AT1R expression in adult Sprague-Dawley proximal tubule and cultured proximal tubule, and this inhibition is mediated via Gs-coupled DA1 receptors. When 3-wk-old animals were given L-DOPA or captopril for 1 wk, MAP was not altered (70 +/- 8 vs. 60 +/- 4 or 61 +/- 5 mmHg), but proximal tubule AT1R mRNA was no longer significantly different between SHR and WKY (68 +/- 9 vs. 38 +/- 7 or 20 +/- 3 vs. 47 +/- 15 cpm per cpm of mutant AT1R per cpm beta-actin x 10(-6)), due to a significant decrease in proximal tubule AT1R expression in SHR (P < 0.005, compared with untreated SHR). Immunoreactive proximal tubule AT1R expression also was increased in 4 wk SHR and was reversed with captopril or L-DOPA treatment. Therefore, these results indicate that young, but not adult, SHR have increased expression of proximal tubule AT1R and that chronic L-DOPA or captopril treatment decreased the elevated AT1R expression to control levels. These results provide further support for an important role of the RAS in the development of hypertension in SHR.A potential role for the renin-angiotensin system (RAS) in the development and/or maintenance of hypertension in the genetic model of rat hypertension, spontaneously hypertensive rats (SHR), has been suggested by studies indicating that treatment of immature animals with angiotensin-converting enzyme (ACE) inhibitors prevents subsequent development of hypertension. Because young SHR also demonstrate RAS-dependent increased sodium retention, we examined proximal tubule type 1 angiotensin II receptor (AT1R) mRNA expression in young (4 wk) or adult (14 wk) SHR compared with age-matched Wistar-Kyoto (WKY) rats. Proximal tubules were isolated by Percoll gradient centrifugation, and AT1R mRNA expression was measured by quantitative reverse transcription-polymerase chain reaction (RT-PCR). At 14 wk, when SHR had established hypertension [mean arterial blood pressure (MAP) of SHR vs. WKY: 145 ± 6 vs. 85 ± 5 mmHg, n = 14-15], there were no differences in proximal tubule AT1R mRNA levels [SHR vs. WKY: 79 ± 14 vs. 72 ± 14 counts/min (cpm) per cpm mutant AT1R per cpm β-actin × 10-6, n = 6; not significant (NS)]. In contrast, in 4 wk SHR, at a time of minimal elevations in blood pressure (MAP: 70 ± 8 vs. 63 ± 3), SHR proximal tubule AT1R mRNA levels were 263 ± 30% that of WKY (143 ± 18 vs. 60 ± 11 cpm per cpm of mutant AT1R per cpm β-actin × 10-6, n = 8; P < 0.005). We have recently shown that chronic ACE inhibition decreases proximal tubule AT1R expression and have also shown that chronicl-3,4-dihydroxyphenylalamine (l-DOPA) administration inhibits AT1R expression in adult Sprague-Dawley proximal tubule and cultured proximal tubule, and this inhibition is mediated via Gs-coupled DA1 receptors. When 3-wk-old animals were given l-DOPA or captopril for 1 wk, MAP was not altered (70 ± 8 vs. 60 ± 4 or 61 ± 5 mmHg), but proximal tubule AT1R mRNA was no longer significantly different between SHR and WKY (68 ± 9 vs. 38 ± 7 or 20 ± 3 vs. 47 ± 15 cpm per cpm of mutant AT1R per cpm β-actin × 10-6), due to a significant decrease in proximal tubule AT1R expression in SHR ( P < 0.005, compared with untreated SHR). Immunoreactive proximal tubule AT1R expression also was increased in 4 wk SHR and was reversed with captopril orl-DOPA treatment. Therefore, these results indicate that young, but not adult, SHR have increased expression of proximal tubule AT1R and that chronic l-DOPA or captopril treatment decreased the elevated AT1R expression to control levels. These results provide further support for an important role of the RAS in the development of hypertension in SHR.