Upregulation of macula densa cyclooxygenase-2 expression is not dependent on glo - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-22 08:12 |只看该作者 |倒序浏览 |打印

作者：Frank Schweda, Martin Kammerl, Charlotte Wagner, Bernhard K. Krämer, and Armin Kurtz作者单位：1 Institut für Physiologie and 2 Klinik and Poliklinik für Innere Medizin II der Universität Regensburg, D-93040 Regensburg, Germany 4 H, O. g2 ^  ?  C  y" k

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      【摘要】
   Although the regulation of cyclooxygenase-2 (COX-2) expression in the kidney cortex has been extensively characterized, the physiological control mechanisms of COX-2 expression at the level of the kidney and at the level of the tubular cells are not well understood. Based on the current hypothesis that tubular salt transport might be a crucial regulator of COX-2 expression, this study aimed to determine the impact of salt delivery to the tubules (glomerular filtration) for the regulation of COX-2 in the kidney cortex in vivo. To this end, glomerular filtration of the right kidney was abrogated by the ligation of the right ureter of male Sprague-Dawley rats. After 1 wk of ligation, the animals were treated with subcutaneous infusions of furosemide (12 mg·kg -1 ·day -1 ) or with a low-salt or a high-salt diet (0.02% wt/wt; 8% wt/wt), and COX-2 as well as renin mRNA expression were determined in the ligated and the nonligated contralateral kidney. During ureteral ligation, hydronephrosis developed with a reduction of medullary mass, while the cortex was preserved. Expressions of the Na-K-2Cl cotransporter isoforms A and B were both reduced in the hydronephrotic cortex to 70 and 35% of the corresponding contralateral intact kidney. Despite the abrogation of glomerular filtration, detected by inulin clearance measurements, renocortical COX-2 mRNA abundance was stimulated by furosemide treatment (3.2-fold) or low-salt diet (2.9-fold) to similar degrees compared with the intact contralateral kidney (2.7-fold for both treatments), whereas a high-salt diet did not significantly suppress COX-2 mRNA in the macula densa region of either kidney. Renin mRNA expression was regulated strictly in parallel in both kidneys, a low-salt diet or furosemide treatment stimulating and a high-salt diet suppressing it. We conclude from these findings that salt delivery to the tubules is not an essential requirement for the upregulation of COX-2 by salt deficiency or by loop diuretics in the rat kidney cortex nor is it for chronic stimulation of renin mRNA expression.
      【关键词】 kidney renin ureteral ligation3 J, T5 V( X- t& J# \$ b
   IT IS WELL ESTABLISHED that the expressions of cyclooxygenase-2 (COX-2) in the cortical thick ascending limb of Henle (TALH) including the macula densa and of renin in the neighboring juxtaglomerular cells are regulated in parallel by a variety of physiologically relevant parameters such as sodium intake ( 13, 21, 36 ), renal perfusion pressure ( 15, 22, 31 ), or circulating levels of ANG II ( 4, 33 ). It has been speculated that the expression of COX-2 in the macula densa triggers the expression and the secretion of renin by the formation of prostanoids ( 12, 14, 28 ), which are direct stimulators of the renin system at the level of juxtaglomerular cells ( 20 ). The functional and molecular pathways controlling COX-2 expression in the TALH and in the macula densa cells at the organ and on the cellular level are not well understood. Some data suggest that the expression of COX-2 might be dependent on the salt transport activity in the way that a reduction of salt transport upregulates COX-2 expression in vitro ( 3, 35 ) and in vivo ( 23 ). Evidence has been provided in vitro that this regulation of COX-2 expression by salt transport involves changes in intracellular chloride concentration ( 3, 35 ) followed by changes in MAP-kinase activity ( 3, 35 ). Such a control of COX-2 expression by salt transport in the TALH would allow regulation of COX-2 expression by changes in glomerular filtration as well as by changes in proximal tubular sodium reabsorption. Such a linkage, in turn, would also provide an intriguing explanation for the regulation of COX-2 expression and finally also for the yet unexplained regulation of renin expression by salt intake, by renal perfusion pressure and also by loop diuretics, which directly inhibit salt transport in the TALH ( 12, 14, 28 ). An essential link in this contention is that the regulation of COX-2 expression in the TALH and in the macula densa is in fact dependent on salt delivery to the tubules, i.e., glomerular filtration, which has not yet been investigated. Our study therefore aimed to determine the relevance of glomerular filtration to renocortical COX-2 and renin expression. For this purpose, we generated rats with a filtering and a nonfiltering kidney, induced by unilateral ureteral ligation. These animals were then subjected to maneuvers known to regulate COX-2 and renin expression such as salt depletion ( 13, 21, 36 ), a high-salt intake ( 2, 36 ), and treatment with a loop diuretic ( 23 ). We found that these maneuvers stimulated COX-2 as well as renin expression in the filtering and in the nonfiltering kidney almost to the same extent, suggesting that glomerular filtration is not an essential requirement for the upregulation of COX-2 and renin expression./ A% W4 C" v7 h# T: }

MATERIALS AND METHODS

Animals. Male Sprague-Dawley rats were used in the experiments. All animal experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and German laws on the protection of animals.  ]2 s- N; ~& _# N% ]
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Induction of nonfiltering kidneys. Rats (160-180 g) were anesthetized using inhalation anesthesia with Servoflurane (Abbott), a small lower abdominal incision was made, and the right ureter was completely ligated near to its entrance into the urinary bladder. After 2 wk, hydronephrosis developed in all animals, as indicated by the hydronephrotis sacs and the rarefaction of inner medullary tissue.
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Nonfiltering status was confirmed by determination of inulin clearance in three rats. For application of inulin (800 mg/kg body wt as priming dose, 300 mg·kg body wt -1 ·h -1 as continuous infusion), a venous catheter was placed in a femoral vein. After an equilibration period of 30 min, three 20-min urine samples were collected via a catheter placed in the urine bladder. In the middle of the 20-min periods, the corresponding blood samples were taken via a catheter in the carotid artery. After the last period, urine from the hydronephrotic sacs was collected. Inulin concentrations in urine and plasma were measured by the Anthron method as described previously ( 23 ).
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Animal groups. One week after induction of hydronephrosis, the rats were divided into four groups. 1 ) Rats were fed a normal-salt diet [0.6% NaCl (wt/wt), Altromin, Lage, Germany] over a period of 7 days ( n = 8). 2 ) Rats were fed a low-salt diet [0.02% NaCl (wt/wt), Ssniff special diets, Soest, Germany] over a period of 7 days ( n = 9). 3 ) Rats were infused with furosemide (12 mg·kg -1 ·day -1 ) via subcutaneously implanted osmotic pumps (model 2ML1, Alza, Palo Alto, CA) for 7 days. These animals had access to tapwater and a salt solution (0.9% NaCl, 0.1% KCl; n = 5). Furosemide stimulates COX-2 and renin gene expression by blockade of the Na-K-2Cl cotransporter (NKCC2), reducing the salt transport rate of macula densa and TALH cells and hereby mimicking a situation of low tubular salt concentration, irrespective of the actual tubular salt load ( 23 ). Free access to salt water was allowed to prevent a major disturbance of body volume and blood pressure. 4 ) Rats were fed a high-salt diet [8% NaCl (wt/wt), Ssniff special diets, Soest] over a period of 7 days ( n = 5).& L. t& w4 ?4 `6 {6 v0 }" w+ J$ |; t

Organ sampling. Animals were killed by decapitation, and the kidneys were removed. Blood samples were taken for determination of plasma renin activity. The kidneys were cut in longitudinal halves. From one of these halves, the cortex was dissected under a stereomicroscope. Pieces of kidney cortex were frozen in liquid nitrogen and stored at -80°C until isolation of total RNA. The second half was used for immunohistochemistry.. ]; U: |+ x$ m: ^2 H% P2 |8 @
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Extraction of RNA. Total RNA was extracted from the cortex basically according to the acid-guanidinium-phenol-chloroform protocol by Chomczynski and Sacchi ( 5 ). RNA pellets were dissolved in diethylpyrocarbonate-treated water, the yield of RNA was quantified by spectroscopy at 260 nm, and samples were placed in aliquots and stored at -80°C until further processing. The quality of extracted RNA was confirmed by the observation of intact 18S and 28S rRNA bands after gel electrophoresis in an ethidium bromide-stained agarose gel.
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RNase protection assays for COX-2, renin, and cytoplasmic-actin mRNA. COX-2, renin, and actin mRNA levels were measured by RNase protection assays. In brief, after linearization and phenol/chloroform purification, the plasmids yielded radiolabeled antisense cRNA transcripts by incubation with SP6 polymerase (Promega) and [ 32 P]GTP (Amersham Pharmacia) according to the Promega riboprobe in vitro transcription protocol; 5 x 10 5 counts/min of the cRNA probes were hybridized with 20 µg of total RNA (renin), 100 µg of total RNA (COX-2), 5 µg of total RNA ( -actin), or 20 µg of tRNA (negative control) at 60°C overnight and were then digested with RNase A/T1 (RT/30 min) and proteinase K (37°C/30 min). After phenol/chloroform extraction and ethanol precipitation, protected fragments (370, 323, and 303 bp in length for COX-2, renin, and actin, respectively) were separated on an 8% polyacrylamide gel. The gel was dried for 2 h, bands were quantitated in a PhosphorImager (Instant Imager 2024, Packard), and autoradiography was performed at 80°C for 1-3 days.

Determination of NKCC2 isoforms by RNase protection assay. mRNA levels of NKCC2 isoforms were measured by RNase protection assay. For detection of NKCC2 A, NKCC2 B, and NKCC2 F mRNAs, antisense RNA probes were generated by in vitro transcription of the plasmid vector pSP73 (Promega-Serva, Heidelberg, Germany) containing PCR-derived cDNAs of the murine NKCC2 isoforms ( 19 ). The 206-bp fragments, amplified by an isoform-specific upstream primer, namely, 5'-tct tct ttc cac cat ggt-3' for NKCC2 A, 5'-cgg ctt agc cgt gac agt-3' for NKCC2 B, and 5'-ggc ctg agc gta gtt gt-3' for NKCC2 F (each binding at position 867-883 bp), and the downstream primer 5'-cgc gga gac tgt cgt gga-3'(binding at 1,055-1,072 bp, homologous for all 3 isoforms), were cloned in a Bam HI/ Eco RI-digested pSP73 vector using standard protocols. Linearization with Hin dIII and in vitro transcription with SP6 RNA polymerase yielded a 260-bp fragment. Hybridization, RNase digestion, phenol/chloroform extraction, and acrylamid electrophoresis were performed as described above.

Determination of plasma renin activity. Plasma renin activity (PRA) was determined using a commercially available radioimmunoassay kit for ANG I (Byk&DiaSorin).% v5 l! l$ b! E4 U
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COX-2 and renin immunoreactivity. After fixation in methyl-Carnoy solution (60% methanol, 30% chloroform, and 10% acetic acid), tissues were dehydrated by bathing in increasing concentrations of methanol, followed by 100% isopropanol. The tissue was embedded in paraffin, and 4-µm sections were cut with a Leitz SM 2000R microtome (Leica Instruments). After deparaffinization, endogenous peroxidase activity was blocked with 3% H 2 O 2 in methanol for 20 min at room temperature. Sections were layered with the primary antibody (COX-2, Santa Cruz Biotechnology, Santa Cruz, CA, dilution 1:100; renin, Dr. C. Wagner, University of Regensburg, Regensburg, Germany, dilution 1:200) and incubated at 4°C overnight. After the addition of the second antibody (dilution 1:200; biotin conjugated, ICN Pharmaceuticals), the sections were incubated with avidin D horseradish peroxidase complex (Vectastain DAB kit, Vector Laboratory) and exposed to 0.1% diaminobenzidine tetrahydrochloride and 0.02% H 2 O 2 as a source of a peroxidase substrate. Each slide was counterstained with hematoxylin. As a negative control, we used the second antibody only without incubation with the primary antibody (16a).5 F4 }2 y; v3 g/ j6 x5 _2 O
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Statistical analysis. Intraindividual differences between the ligated and the unligated kidney [NKCC2 expression, glomerular filtration rate (GFR)] were analyzed by Student's paired t -test. Comparisons between the control group and the low-salt or the furosemide group (COX-2 mRNA, renin mRNA, PRA) were made by ANOVA and Bonferroni adjustment for multiple testing. P values 4 B" F- {( V$ r# j

RESULTS

Complete right renal ureteral ligation was performed for 2 wk, during which the kidneys developed hydronephrosis with fluid retention of 3.71 ± 0.79 ml ( n = 8) per kidney. As a consequence, the inner medulla showed clear signs of mass reduction. In the cortex and the outer medulla of the hydronephrotic kidney, tubuli were dilated ( Fig. 1 ). To verify the absence of glomerular filtration, inulin clearance was determined in three hydronephrotic rats. On average, the inulin clearance amounted to 0.59 ± 0.163 ml/min. However, no inulin could be detected in the hydronephrotic sacs, indicating that GFR in the hydronephrotic kidneys was zero ( Fig. 2 ).

Fig. 1. Histology of a hydronephrotic kidney ( A ) and the contralateral intact counterpart ( B; x 50). Tubuli are markedly dilated in the cortex and the outer medulla of the hydronephrotic kidney. Immunostaining for renin shows typical localization of renin in the afferent arterioles.
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Fig. 2. Glomerular filtration rate (GFR) of kidneys with ureteral ligation (UL) compared with the contralateral intact kidneys. Values are means ± SE of 3 kidneys each.
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To exclude the possibility that the abrogation of glomerular filtration and the concomitant reduction of tubular salt load might result in a counterregulatory induction of tubular salt transporters, we determined the mRNA expression of NKCC2 in the cortex of hydronephrotic and control kidneys of eight rats on a normal-salt diet. Expression of NKCC2 A and NKCC2 B, isoforms of NKCC2 that have been demonstrated to be expressed in the macula densaand the neighboring cells of the TALH ( 34 ), was downregulated in the hydronephrotic kidneys to 70% (NKCC2 A, P

Fig. 3. mRNA expression of Na-K-2Cl cotransporter (NKCC2) isoforms A, B, and F in the cortex of hydronephrotic and intact kidney cortices. Values are means ± SE. * P

Treatment of the rats with a low-salt diet or furosemide treatment over 1 wk increased PRA significantly, whereas a high-salt diet suppressed PRA, indicating sufficient stimulation protocols ( Fig. 4 ). Notably, the stimulation of PRA was more pronounced in the furosemide-treated animals compared with the low-salt diet, which is in line with the results obtained in normal rats ( 2 ).( T+ S3 x" K; n8 R+ U( {# A# `

Fig. 4. Effects of high-salt diet, low-salt diet, and furosemide treatment on plasma renin activity of rats with unilateral hydronephrosis. Values are means ± SE. * P

As demonstrated previously, a low-salt diet resulted in a significant increase in COX-2 mRNA abundance in the cortex of the intact kidney (2.7-fold of control; Fig. 5, top ). Abrogation of glomerular filtration by ureteral ligation did not critically affect the stimulation of COX-2 expression by low-salt intake as COX-2 mRNA abundance was enhanced to a similar degree in the hydronephrotic kidneys as in the intact kidneys (2.7-fold of control in the unligated, filtering kidney; 2.9-fold of control in the ligated, nonfiltering kidney; Fig. 5, top ). Treatment of the rats with a high-salt diet over 1 wk tended to decrease COX-2 mRNA levels in both the ligated as well the intact kidney (0.6-fold of control for both, n = 5). However, this change did not reach statistical significance in either kidney ( Fig. 5, top ). Furthermore, the regulation of renin synthesis by salt intake was unaffected by ureteral ligation, as renin mRNA expression was stimulated to 2.2-fold of control in the nonfiltering as well as in the filtering kidneys by a low-salt diet, whereas a high-salt diet significantly suppressed renin mRNA expression in both kidneys ( Fig. 5, bottom ).) e/ ^  c& r4 \' w6 E
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Fig. 5. Cyclooxygenase-2 (COX-2) mRNA ( top ) and renin mRNA ( bottom ) levels in kidneys with chronic ureteral occlusion and in contralateral intact kidneys of rats kept on a normal-salt diet (control), of rats treated with a high-salt diet and a low-salt diet, and of rats subcutaneously infused with furosemide. Values are means ± SE. * P

To obtain information about the intracortical distribution of COX-2 protein expression, paraffin sections of ligated kidneys were stained for COX-2 immunoreactivity. As shown in Fig. 6, the localization of COX-2 immunoreactivity was mainly restricted to the macula densa region of the hydronephrotic kidney under baseline as well as under stimulated conditions, indicating that no atypical COX-2 expression was accountable for the increase in COX-2 abundance. Similarly, renin immunostaining was only found in the juxtaglomerular cell region of the afferent arterioles ( Fig. 1 ).' w- r+ D' u5 Z' I
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Fig. 6. COX-2 immunoreactivity in the cortex of a rat kidney with ureteral occlusion under control conditions ( A ) and under a low-salt intake over 1 wk ( B; magnification x 200)." f# |3 G& I% ?% G+ X% F/ F
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In parallel with the mRNA, COX-2 protein immunoreactivity, semiquantitated as the percentage of immunopositive glomeruli, increased from 3.7 ± 0.9 to 17.1 ± 0.7% positive glomeruli in the unligated kidneys and from 3.2 ± 0.7 to 15.3 ± 2.7% in hydronephrotic cotices under a low-salt diet, whereas a high-salt diet only tended to decrease COX-2 immunoreactivity in the intact (1.7 ± 0.8%) as well as in the nonfiltering kidney (2.0 ± 0.7%; Fig. 7 ).
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Fig. 7. Relative number of glomeruli positively stained for COX-2 in hydronephrotic and intact kidneys under control conditions, under a low-salt diet, and under furosemide treatment. Values are means ± SE. * P
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Pharmacological inhibition of the NKCC2 in the TALH and the macula densa cells was achieved by subcutaneous infusions of furosemide. As the salt transport rate of NKCC2 has been demonstrated to be crucial in the salt-dependent regulation of renin release ( 16 ), blockade of this transporter mimics a low-salt condition at the macula densa site. To avoid detrimental salt and water loss in these animals, they had free access to salt water, normal tapwater, and to normal food. Furosemide clearly upregulated COX-2 mRNA in the intact kidney and in the hydronephrotic kidney to 2.7- and 3.2-fold of the respective control value ( Fig. 5 ). Again, not only COX-2 but also renin mRNA expression were stimulated to similar degrees in the intact and in the ligated kidney ( Fig. 5 ), and no atypical localization of COX-2 was detected immunohistochemically.! C$ p* D+ A8 G' v+ X! ^
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DISCUSSION! M% f3 [- Z$ h  H+ i

COX-2 is constitutively expressed in the tubular cells of the TALH including the macula densa region. COX-2 mRNA expression has been found to be regulated in parallel to the renin system with a high-salt intake suppressing COX-2 and renin gene expression and with a low-salt diet or furosemide treatment stimulating it ( 13, 21, 36 ). Based on the fact that prostaglandins stimulate renin release and renin synthesis, and a pharmacological blockade or genetic deletion of COX-2 attenuated or even blunted the stimulation of renin release by a low-salt diet or furosemide treatment, a concept of salt-dependent regulation of the renin system was developed that considered COX-2-derived prostaglandins to be the mediator of the macula densa signal toward the renin-producing juxtaglomerular cells ( 12 ). This concept is markedly strengthened by elegant in vitro studies demonstrating that the release of prostaglandin E 2 by macula densa cells is indeed stimulated by low chloride concentration at the macula densa site ( 35 ) and, moreover, that low chloride concentration stimulates COX-2 mRNA expression in cultured cells of the TALH as well as in macula densa cells ( 3, 35 ), suggesting that COX-2 gene expression and COX-2 activity are controlled by the salt transport of these tubular cells. However, so far it has not been proven that salt delivery to the tubules is a prerequisite for the regulation of COX-2 gene expression by salt intake. Therefore, in the present study, we tested the hypothesis that the regulation of COX-2 gene expression in the macula densa cells by different salt diets is prevented by an abrogation of glomerular filtration and tubular salt load. Interestingly, we did not find any significant differences in the regulation of COX-2 or renin mRNA expression by a low-salt diet, a pharmacological blockade of NKCC2, or a high-salt diet between intact, filtering kidneys and hydronephrotic, nonfiltering kidneys. Therefore, our study for the first time provides evidence that changes in tubular salt load are not required for the salt-dependent regulation of COX-2 gene expression and, moreover, we demonstrate that neither salt-dependent regulation of renin mRNA expression is dependent on glomerular filtration. Consequently, our data strongly argue against the hypothesis that chronic changes in the bodys salt intake are functionally linked to COX-2 or renin gene expression by changes in the tubular salt load and subsequent alterations of the salt transport rate of the macula densa cells.: x( @3 A6 D1 g! @( C$ K/ o& M. A; k  O" Q

To produce nonfiltering kidneys, we performed ureteral ligation hereby inducing hydronephrosis. Although the kidney cortex was structurally well preserved even after 2 wk of complete ureteral ligation, the hydronephrosis model is a rather complex model with which to study the cortical expression of COX-2 as ureteral ligation presumably induces inflammation of the obstructed kidney that goes along with upregulation of renal COX-2 activity. However, to date, it is not entirely clear which renal structures are the site of COX-2 upregulation. COX-2 expression is clearly enhanced in obstructed human ureters ( 25 ). In rats, ureteral ligation for 2 wk did not change macula densa COX-2 expression but induced some diffuse immunoreactivity in tubular structures and interstitial cells of the hydronephrotic kidneys ( 24 ). As it is not reported whether the additional staining is located in the cortex, that study might be in some contrast to our findings as we do not find any atypical localization of COX-2 in the hydronephrotic kidney cortex, while we also observed no differences of COX-2 expression in the macula densa between the hydronephrotic and the intact kidney. Ureteral ligation for 1 day significantly reduced the cortical COX-2 mRNA abundance without changing the distribution of COX-2 within the cortex ( 6 ), whereas COX-2 mRNA abundance was elevated in the outer and inner medulla of the obstructed kidney ( 6 ). These morphological studies are well paralleled by several functional investigations. Thus PGE 2 release from isolated, perfused kidneys of rabbits is similar in hydronephrotic and normal kidneys under baseline conditions, whereas the stimulation of PGE 2 release by bradykinin was enhanced in the hydronephrotic kidneys ( 29 ). A more detailed analysis of the intrarenal distribution of PGE 2 formation revealed that the renal medulla and not the cortex is the site of enhanced PGE 2 synthesis in obstructed kidneys ( 32 ) and that PGE 2 formation of glomeruli of kidneys with ureteral ligation is lower than in their unaffected counterparts ( 11 ). Taken together, ureteral ligation impacts COX-2 expression in the kidney as it clearly stimulates COX-2 expression and PGE 2 formation in the medulla and the ureter, whereas it has no major effect or even inhibits these parameters in the cortex. This conclusion is perfectly in line with our results, as we found COX-2 expression in the cortex to be restricted to the macula densa region and a somewhat lower COX-2 mRNA abundance in the hydronephrotic kidneys compared with their intact counterparts. However, as already mentioned, because COX-2 expression in the macula densa region might be influenced by other mechanisms besides salt delivery to the tubules, putative differences in absolute COX-2 expression levels between the hydronephrotic and the intact kidney are hardly attributable to a single factor. Therefore, the focus of our study was to compare the stimulability of COX-2 by low-salt or furosemide treatment as well as the suppression of COX-2 by a high-salt diet in hydronephrotic and normal kidneys.
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We demonstrate that glomerular filtration in the ligated kidneys had completely ceased as indicated by the absence of inulin filtration into the hydronephrotic sac. Moreover, mRNA expression of NKCC2 isoforms A and B, both known to be expressed in the TALH including the macula densa region ( 34 ), was significantly downregulated in the hydronephrotic kidneys. However, despite the blunted salt delivery to the macula densa, COX-2 expression was stimulated by a low-salt diet or furosemide treatment to similar extents in the hydronephrotic and the intact contralateral kidney as indicated by cortical COX-2 mRNA expression and the relative number of COX-2-positive-stained glomeruli. As it is very unlikely that these treatments have a major influence on the inflammation activity of the hydronephrotic kidney, we infer from these data that neither glomerular filtration as such nor changes in glomerular filtration are prerequisites for the regulation of COX-2 expression in the TALH and in the macula densa.

In parallel with COX-2 expression, a low-salt diet or furosemide treatment stimulated renin expression in the obstructed kidney to the same extent as in the intact kidney. This result is in good accordance with previous studies. It is known that acute ureteral occlusion causes rapid activation of the renin system ( 27 ) that keeps maintained in the chronic phase, if ureteral occlusion is performed in the neonate rat ( 9 ), but returns to normal values if ureteral occlusion is performed in the adult rat ( 10, 26 ). Moreover, the chronic stimulation of the renin synthesis by low-salt intake, reduced perfusion pressure or ACE inhibition was preserved in hydronephrotic kidneys of mice ( 1, 37, 38 ), whereas a high-salt intake suppressed renin content in both the intact and the hydronephrotic kidneys ( 38 ), which is also in line with our results. Although renin synthesis was not altered by ureteral ligation in those studies, renin release was markedly affected by this maneuver ( 38 ), underlining the major role of tubular salt load on the acute regulation of the renin secretion ( 16, 30 ).1 Y2 Q' L7 A9 R+ [  O9 N1 K* l8 w

In principle, the conclusion that glomerular filtration is not indispensably required for salt-dependent regulation of COX-2 and renin synthesis does not necessarily exclude that tubular salt transport is relevant for the regulation of the expression of these two enzymes, because it is conceivable that even in nonfiltering, hydronephrotic kidneys a recirculating tubular salt transport can occur. In this case, furosemide could still act at the cellular level by inhibiting transcellular salt transport by inhibiting NKCC2. We consider such a mechanism, however, as less likely, because one would expect that the recirculating transepithelial salt transport rate in hydronephrotic kidneys should be lower than the tubular salt transport in intact, filtering kidneys. In fact, it has already been shown that ureteral occlusion leads to a rapid decline of salt transport by the TALH ( 18 ), which is compatible with the reduced expression of NKCC2 in our study. A reduction of tubular salt transport should increase basal COX-2 expression and at the same time attenuate the stimulation of COX-2 expression by maneuvers inhibiting tubular salt transport. Both predictions could not be verified in our study. It seems therefore more likely that the stimulation of tubular COX-2 expression in vivo by loop diuretics and by low-salt intake could be indirectly mediated rather than be due to direct effects of the drugs on tubular transport. A common indirect mediator of both maneuvers could be reductions in the extracellular volume, which are sensitively registered by cardiac and thoracic blood volume sensors. Although so far we found only trends for a shrinkage of extracellular volume in our model of chronic furosemide infusion ( 23 ), we have to assume that a very sensitive and precise measuring of the extracellular volume exists that directs the animals to drink the enormous amounts of salt water to compensate for renal salt and water loss. In this view, the stimulation of the renin system in these animals may be taken as a further indication for a minute volume deficit. The existence of an interaction between cardiac volume sensors with the renin system and with the renal tubular function is well established ( 7 ). The molecular pathways involved in these interactions, however, are not well understood. An obvious mediator function could be provided by renal nerve activity ( 8 ). Our previous data, however, show that the stimulations of renocortical COX-2 and renin expression by furosemide and by low salt combined with ACE inhibition are not influenced by effective renal denervation or -adrenoreceptor blockade ( 17 ). Thus the mechanisms, by which volume changes could trigger tubular COX-2 expression and juxtaglomerular renin expression, still remain elusive.; q5 t9 |3 T$ y; p1 V

Taken together, our findings suggest that glomerular filtration and, in consequence, also tubular salt transport are not a general indispensable requirement for the regulation of COX-2 in the TALH and in the macula densa region and of renin in the juxtaglomerular cells of rat kidneys by salt deficiency and by loop diuretics.: U* X- w  t- {  V

GRANTS& _! P4 K6 r' O$ |

The study was supported by a grant from the Deutsche Forschungsgemeinschaft (KU859/12-2; SCHW778/2-1).0 m7 H  a! e+ `0 W2 V
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ACKNOWLEDGMENTS
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The expert technical assistance provided by M. Hamann, S. Lucas, and M. Ivanjak is acknowledged.
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Castrop H, Klar J, Wagner C, Hoecherl K, and Kurtz A. General inhibition of renocortical cyclooxygenase-2 expression by the renin-angiotensin system. Am J Physiol Renal Physiol 284: F518-F524, 2003." R+ `0 i9 V. {  Y! D2 [! I( d# M, O1 `& e
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Cheng HF, Wang JL, Zhang MZ, McKanna JA, and Harris RC. Role of p38 in the regulation of renal cortical cyclooxygenase-2 expression by extracellular chloride. J Clin Invest 106: 681-688, 2000.% N2 ^  Z1 f5 T* @
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Cheng HF, Wang JL, Zhang MZ, Miyazaki Y, Ichikawa I, McKanna JA, and Harris RC. Angiotensin II attenuates renal cortical cyclooxygenase-2 expresssion. J Clin Invest 103: 953-961, 1999.6 k8 W9 V9 G/ M" }' s# U
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