Neonatal RAS inhibition changes the phenotype of the developing thick ascending limb of Henle

【摘要】  Pharmacological interruption of angiotensin II type 1 (AT 1 ) receptor signaling during nephrogenesis in rats perturbs renal tubular development. Perturbed tubulogenesis may contribute to long-term impairment of urinary concentrating ability, which is the main functional irreversible defect. The aim of this study was to further characterize tubular developmental deficits in neonatal rats, focusing on the thick ascending limb of Henle (TALH), known to undergo profound developmental changes and to be involved in urine-concentrating mechanisms. We have carried out immunohistochemistry and Western immunoblotting using antibodies directed against the major histocompatibility complex class II (MHC II) molecule and different TALH-specific markers, namely, cyclooxygenase-2 (COX-2), Tamm-Horsfall glycoprotein (THP), and the bumetanide-sensitive Na + -K + -2Cl - cotransporter (BSC-1/NKCC2). Immunohistochemistry demonstrated expression of MHC II, COX-2, THP, and BSC-1/NKCC2 proteins in normally developing TALH cells. The AT 1 -receptor antagonist losartan abolished MHC II expression exclusively in the developing TALH cells. Increased expression of COX-2 and THP was observed in the TALH cells of losartan-treated rats. Western immunoblotting confirmed increases in cortical and medullary COX-2 and THP abundance and revealed a decrease in cortical BSC-1/NKCC2 abundance in response to losartan treatment. We conclude that neonatal losartan treatment causes significant changes in the phenotype of the developing TALH in the rat.

【关键词】  renal development AT receptor blockade major histocompatibility complex class II molecule cyclooxygenase TammHorsfall glycoprotein bumetanidesensitive sodiumpotassiumchloride cotransporter

ANGIOTENSIN II (ANG II) has important effects on structural development and functional differentiation of the kidney in the perinatal life of many species. Pharmacological or genetic inhibition of ANG II or the angiotensin II type 1 (AT 1 ) receptor during the perinatal period induces renal developmental defects. A main functional finding in adult rats subjected to neonatal inhibition of the renin-angiotensin system (RAS) is impaired urinary concentrating ability, which is causally related to medullary atrophy and tubulointerstitial abnormalities ( 6, 7 ). ANG II may be pivotal for growth and differentiation of tubular and interstitial cells, given its growth-stimulating effects on these cells in vitro ( 18, 40, 41 ). Indeed, pharmacological inhibition of ANG II in neonatal rats perturbs medullary tubulogenesis and interferes with the development of interstitial tissue ( 16 ). One postnatal change occurring in the tubulointerstitium is the acquisition of the major histocompatibility complex class II (MHC II) molecule ( 34 ). MHC II expression in tubular epithelium increases with maturation ( 22, 34 ) and may participate in histogenesis and morphogenesis ( 37 ). The primary aims of the present study were to examine 1 ) the temporal and spatial changes in renal MHC II expression after neonatal losartan treatment; 2 ) whether the losartan effect is direct or may be related to hemodynamic changes, by comparing losartan with nifedipine (a calcium channel blocker); and 3 ) whether the losartan effect on MHC II expression is specific to the developing kidney. Although other organs, such as intestines, lungs, and liver, undergo postnatal development ( 2, 26, 28, 43 ), express AT 1 receptors ( 29, 31, 36 ), and upregulate MHC II ( 22 ), only in the kidney are irreversible developmental defects found after interrupted neonatal AT 1 receptor signaling ( 8 ). Our data have shown that losartan abolished MHC II expression exclusively in epithelial cells of the developing thick ascending limb of Henle (TALH), raising the hypothesis of specific changes in the TALH phenotype after neonatal RAS inhibition.

The TALH has an important role in NaCl reabsorption and urine concentration by generating a high osmolality in the renal medulla through the countercurrent multiplier ( 11 ). Because losartan-induced changes in the developing TALH could underlie long-term urine-concentrating disability in adult rats ( 6 ), the secondary aims of the present study were to further characterize the changes in the developing TALH after neonatal AT 1 -receptor antagonism. We examined the abundance of 1 ) Na + -K + -2Cl - cotransporter [rat type 1 bumetanide-sensitive cotransporter (BSC-1/NKCC2)], one of the major transporters involved in salt reabsorption by TALH cells ( 15 ); 2 ) cyclooxygenase-2 (COX-2), a TALH marker that is transiently upregulated in the developing kidney and plays an important role in differentiation and early function of developing nephrons ( 38, 44 ); and 3 ) Tamm-Horsfall glycoprotein (THP), a unique protein that is produced exclusively by TALH cells and is thought to be linked to the function of the TALH ( 30 ).

MATERIALS AND METHODS

General procedures. Female Wistar rats (Möllegaard Breeding Centre, Copenhagen, Denmark) were transported to our facility on days 14-15 of pregnancy. They were observed carefully for determination of the day of delivery. Both male and female pups were included in the study. Weight-matched pups were divided into three groups receiving daily intraperitoneal injections of either the AT 1 blocker losartan (2 x 15 mg/kg), calcium channel blocker nifedipine (2 x 2, 5 mg/kg), or isotonic saline vehicle (2 x 10 ml/kg) from postnatal day 0 to day 12. Rats had free access to standard rat chow and tap water and throughout the study were kept in rooms with a controlled temperature of 24°C and a 12:12-h light-dark cycle. All experiments were approved by the local ethics committee in Gothenburg.

Protocol. Pups ( n = 6-7/group) were killed by decapitation on postnatal days 2, 4, 9, and 13 and had their kidneys, small intestine, liver, lungs, and spleen rapidly removed. All organ specimens were embedded in Tissue-Tek OCT compound and snap-frozen in liquid nitrogen. Five-micrometer-thick sections were mounted on Superfrost Plus glass slides and kept at -20°C until used for immunohistochemistry.

In separate experiments, rats were treated postnatally with losartan or saline vehicle ( n = 8-9/group) for 4 or 9 days before death. One kidney was dissected into medulla and cortex, snap-frozen in liquid nitrogen, and used for Western immunoblot analysis. The other kidney was fixed in formalin, embedded in paraffin, and used for immunohistochemistry.

Immunohistochemistry. Immunostaining for MHC II was performed on frozen sections from kidneys, lungs, liver, and intestines. Immunostaining for COX-2 and BSC-1/NKCC2 was performed on frozen kidney sections. Immunostaining for THP was performed on both frozen and paraffin sections.

The sections were incubated with 40 µl of primary antibodies ( Table 1 ) for 1 h in a humid atmosphere at room temperature. After depletion of endogenous peroxidase activity with 0.3% H 2 O 2, sections were incubated with relevant secondary antibodies ( Table 1 ) for 45 min. All antibodies were linked to horseradish peroxidase. Binding of peroxidase-labeled secondary antibodies was detected by incubation with substrate 3-amino-9-ethyl-carbazole (Sigma, St. Louis, MO) containing H 2 O 2, yielding a brown stain. Counterstaining with Mayer's hematoxylin was performed only on the sections stained for MHC II. Spleen specimens were used as a positive control for MHC II expression. Negative control samples were obtained by replacement of primary antibodies with 1% BSA.

Table 1. Primary and secondary antibodies

To examine whether the tubules expressing MHC II or COX-2 were TALH, MHC II or COX-2 immunostaining, as described above, was followed by THP ( 13 ) staining. Binding of peroxidase-labeled secondary antibody was detected by incubation with substrate Vector SG (Vector Laboratories), yielding a blue-grey stain. To intensify the contrast between brown and blue-grey stains, no counterstaining with Mayer's hematoxylin was performed.

Immunostaining for THP was performed on paraffin sections. Sections were heated at 60°C for 30 min, deparaffinized, and boiled in citric acid buffer (0.01 M, pH 6.0) for 30 min. Nonspecific binding was blocked by incubation with nonimmune donkey serum (15 min). After overnight incubation (at 4°C) with anti-THP antibody, sections were depleted of endogenous peroxidase activity and incubated with relevant secondary antibody ( Table 1 ). Immunoreactivity was visualized using 3-amino-9-ethyl-carbazole, and counterstaining was performed with Mayer's hematoxylin.

All antibodies used for immunohistochemistry were diluted in phosphate-buffered saline containing 1% of BSA.

Western immunoblotting. Dissected day 4 and day 9 renal cortexes and medullas were homogenized in buffer containing 250 mM sucrose, 10 mM HEPES-Tris (pH 6.95), and protease inhibitors (Complete Mini, Roche). Protein concentration was measured using a Bio-Rad Protein Assay kit. Aliquots of 100 µg of protein were solubilized in Laemmli sample buffer and separated, under reducing conditions, by electrophoresis on 4-15% Tris·HCl gradient gels (Bio-Rad, Hercules, CA). Proteins were transferred to a polyvinylidene difluoride membrane (Amersham, Uppsala, Sweden). After being blocked in 5% nonfat milk with phosphate-buffered saline plus 0.1% Tween 20 (PBS-T), the membrane was incubated with primary anti-COX-2 (1:100), anti-THP (1:100), or anti-BSC-1/NKCC2 (1:500) antibodies diluted in 5% nonfat dry milk in PBS-T for 1 h. The proteins were detected using horseradish peroxidase-linked donkey anti-goat IgG, donkey anti-sheep IgG, or goat anti-rabbit IgG, respectively (1:1,000, 30-min incubation; Santa Cruz Biotechnology, Santa Cruz, CA), and the ECL detection system (Amersham). The specificity of anti-COX-2 (sc-1747) antibody was shown by its preabsorption with an excess of specific blocking peptide (sc-1747 P; Santa Cruz Biotechnology), and the specificity of other antibodies was shown by the omission of primary antibody. Bands were visualized using a Fuji LAS-1000 cooled charge-coupled device camera/Dark Box, employing Image Reader LAS-1000 v1.1 software, and the density of the bands was analyzed with the help of Image Gauge software v3.45. -Actin was used as an internal control, and the level of COX-2, THP, and BSC-1/NKCC2 was expressed in relation to -actin.

Statistical analysis. All values are expressed as means ± SD. Statistical comparisons were made according to a two-tailed Student's t -test for unpaired data. A P value <0.05 was considered statistically significant.

RESULTS

Effect of losartan on MHC II expression in neonatal kidneys and other organs. In control kidneys, MHC II staining was already evident at postnatal day 2, and it localized mainly to the tubules of medullary rays, corresponding to the position of the TALH ( Fig. 1, A - C ). The interstitium was stained only occasionally ( Fig. 1, A and B ). This pattern persisted until day 13, when additional proximal tubules and interstitial cells became positive for MHC II ( Fig. 1, G - I and M - O; pictures from day 13 are not shown). Double immunostaining with anti-MHC II and anti-THP antibodies confirmed that MHC II-positive tubules were TALH tubules ( Fig. 2 A ).

Fig. 1. A - U : major histocompatibility complex class II (MHC II) molecule expression in developing rat kidneys. Kidney sections from 2 ( A - F )-, 4 ( G - L )-, and 9-day-old rats ( M - U ) treated with saline vehicle ( A - C, G - I, and M - O ), losartan ( D - F, J - L, and P - R ), or nifedipine ( S-U ) are shown. The losartan-treated rat is devoid of MHC II staining in developing cortical and medullary tubules. A few MHC II-positive cells are visible in the interstitium of losartan-treated rats ( F, K, L, Q, and R ). Nifedipine-treated rat demonstrates MHC II staining compatible with that seen in saline-treated rats. Magnification is indicated by the scale bars.

Fig. 2. A and B : double [MHC II and Tamm-Horsfall glycoprotein (THP)] immunostaining of kidney sections from 9-day-old rats treated with saline (Sal) vehicle or losartan (Los), respectively. A : MHC II (brown stain) is expressed by THP-positive (blue-grey stain) cells from thick ascending limb of Henle (TALH) in Sal-treated rat. B : Los-treated rat demonstrates no MHC II expression in TALH cells. Magnification is indicated by the scale bars.

In losartan-subjected kidneys, TALH staining for MHC II was absent at all time points investigated ( Figs. 1, D - F, J - L, and P - R, and 2 B ). Conversely, frequent interstitial MHC II-positive cells were found in 9 ( Fig. 1, Q and R )- and 13-day-old (data not shown) rats treated with losartan. In 9-day-old rats subjected to nifedipine treatment, the renal pattern of MHC II expression was comparable to that observed in saline-treated rats ( Fig. 1, S - U ). Preserved MHC II expression by TALH cells and normal renal morphology (data not shown) of nifedipine-treated neonatal rats prevented us from further analyses of kidneys from rats treated neonatally with nifedipine. Control and losartan-treated rats demonstrated no differences in MHC II expression in the intestines, liver, and lungs ( day 9 ) ( Fig. 3, A - F ).

Fig. 3. A - F : MHC II expression in intestines ( A and B ), liver ( C and D ), and lungs ( E and F ) from 9-day-old rats treated with Sal vehicle or Los. No differences in MHC II expression are observed between Sal- and Los-treated rats. Magnification is indicated by the scale bars.

To further characterize losartan-induced changes in TALH cells and determine whether the demonstrated decrease in MHC II expression in TALH cells is selective for MHC II protein or is a part of a generalized decrease in the TALH cell pool, we carried out semiquantitative immunoblotting and immunohistochemistry for three specific markers of TALH cells: COX-2, THP, and BSC-1/NKCC2.

Effect of losartan on COX-2 expression in neonatal kidneys. The antibody to COX-2 of the TALH revealed a band at 72 kDa. The band disappeared when the COX-2 antibody was preabsorbed with an excess of specific blocking peptide ( Fig. 4 A ). The abundance of the COX-2 protein did not differ in the cortex and medulla of control and losartan-treated rats at day 4. The abundance of the COX-2 protein in the cortex and medulla of losartan-treated rats was significantly increased at day 9 ( Fig. 4, A and B ).

Fig. 4. A - F : expression of cyclooxygenase-2 (COX-2) protein in the kidneys from 4- and 9-day-old neonatal rats treated with Sal vehicle or Los. A : representative Western blot showing a 72-kDa band that was recognized by anti-COX-2 antibody. Neg., negative control with primary antibody being preabsorbed with an excess of specific blocking peptide. B : densitometric analysis of the 72-kDa bands showed a significant increase in the level of COX-2 expression in the cortex and medulla from 9-day-old rats treated with Los. Values are means ± SD ( n = 8-9/group). * P < 0.05 vs. Sal vehicle. C - F : COX-2 immunostaining is present in the kidneys from 9-day-old rats in both groups (brown stain). COX-2 labeling is increased in Los-treated rats ( D ), and it is confined to the TALH cells, as revealed from the colocalization with THP (blue-grey stain; F ). Magnification is indicated by the scale bars.

Immunocytochemical staining showed that the immunopositive signal for COX-2 was present in TALH cells from control and losartan-treated kidneys (brown stain). In control kidneys, COX-2 immunolabeling was restricted mainly to cortical TALH cells, with a few positive cells being present in medullary TALH ( Fig. 4, C and E ), as seen from the colocalization with THP (blue-grey stain). In the losartan-subjected kidneys, a marked increase in the number of COX-2-positive medullary TALH cells was observed ( Fig. 4, D and F ).

Effect of losartan on THP expression in neonatal kidneys. The antibody to THP of the TALH revealed a band at 100 kDa. The band disappeared when the primary antibody was omitted ( Fig. 5 A ). The abundance of THP protein did not differ in the cortex and medulla of control and losartan-treated rats at day 4. The abundance of THP protein in the cortex and medulla from losartan-treated rats was increased at day 9 ( Fig. 5, A and B ).

Fig. 5. A - F : expression of THP protein in the kidneys from 4- and 9-day-old neonatal rats treated with Sal vehicle or Los. A : representative Western blot showing a 100-kDa band that was recognized by anti-THP antibody. B : densitometric analysis of the 100-kDa bands showed a significant increase in THP expression in the cortex and medulla from 9-day-old rats treated with Los. Values are means ± SD ( n = 8-9/group). * P < 0.05 vs. Sal vehicle. THP immunostaining is present in the cortex ( C and D ) and medulla ( E and F ) from 9-day-old rats in both groups. Los-treated rat demonstrates increased THP labeling in the cortex ( D ) and medulla ( F ). Arrow in D depicts the THP protein in the dilated TALH lumen. Magnification is indicated by the scale bars.

Immunocytochemical staining showed that the signal for THP was present in cortical and medullary TALH cells from control and losartan-treated kidneys ( Fig. 5, C - F ). Losartan-subjected kidneys demonstrated much heavier labeling for THP. Notably, the THP-positive signal was found in the dilated TALH lumen from losartan-subjected kidneys ( Fig. 5, D and F ).

Effect of losartan on BSC-1/NKCC2 expression in neonatal kidneys. The antibody to BSC-1/NKCC2 of the TALH-labeled two bands: one band corresponded to a size of 160 kDa and the other to 320 kDa ( Fig. 6 A ). These two bands probably represent the monomer (160 kDa) and dimer (320 kDa) of the cotransporter ( 17 ). The abundance of the BSC-1/NKCC2 dimer (320-kDa band) in the cortex of losartan-subjected kidneys was decreased at days 4 and 9, but there were no differences in the abundance of BSC-1/NKCC2 monomer (160 kDa) between control and losartan-treated rats ( Fig. 6, A and C ). The sum of both the 320- and 160-kDa BSC-1/NKCC2 bands was significantly decreased in the cortex of losartan-subjected kidneys compared with controls at both days 4 and 9 ( Fig. 6, B and C ). The abundance of the 320- and 160-kDa bands did not differ in the medulla of control and losartan-treated rats ( Fig. 6, A - C ). Immunocytochemical staining showed that BSC-1/NKCC2 was present at the apical plasma membrane of cortical TALH and medullary TALH ( Fig. 6, D - I ).

Fig. 6. A - I : expression of bumetanide-sensitive Na + -K + -2Cl - cotransporter (BSC-1/NKCC2) protein in the kidneys from 4- and 9-day-old neonatal rats treated with Sal vehicle or Los. A : 2 bands (320 and 160 kDa) were recognized by anti-BSC-1/NKCC2 antibody. B and C : densitometric analysis of 320- and 160-kDa bands showed a significant decrease in the expression of BSC-1/NKCC2 in the cortex, but not the medulla, from 4- and 9-day-old rats treated with Los. Values are means ± SD ( n = 8-9/group). * P < 0.05 vs. Sal vehicle. BSC-1/NKCC2 immunostaining is present in the cortex ( D - G ) and medulla ( H and I ) from 9-day-old rats in both groups. Magnification is indicated by the scale bars.

DISCUSSION

The present study demonstrates the changes in the phenotype of developing medullary and cortical TALH caused by AT 1 -receptor inhibition in neonatal rats. These changes are characterized by downregulation of MHC II and BSC-1/NKCC2 and upregulation of COX-2 and THP.

MHC II expression by TALH cells is subject to normal developmental regulation and is sustained over the period of nephrogenesis ( 34 ), which continues into the second postnatal week in the rat ( 35 ). This raises an exciting possibility of a role in the genesis of the TALH. It is noteworthy that prominent postnatal changes, comprising TALH cell proliferation and transformation, together with the growth and descent of the loops of Henle, occur in and around the corticomedullary area, which is a major growth zone for medullary structures ( 3 ). The engagement of MHC II at the cell surface, at least of immune cells, may regulate cell proliferation ( 1 ). Therefore, we hypothesize that MHC II could be linked to ANG II-induced proliferation of TALH cells, which possess AT 1 receptors and proliferate in response to ANG II ( 41 ).

In the rat, other organs, such as the intestines, lungs, and liver, undergo postnatal development ( 2, 26, 28, 43 ), express AT 1 receptors ( 29, 31, 36 ), and upregulate MHC II ( 22 ). However, the present study shows that MHC II expression in other postnatally developing organs is unaffected by AT 1 -receptor inhibition, pointing to a specific association between the RAS and MHC II in the developing TALH.

The loss of MHC II protein in losartan-subjected kidneys corroborates our recent finding of the suppression of a few genes encoding different members of the MHC family in the neonatal medulla of AT 1 receptor-inhibited rats ( 4 ). This may be due to a losartan-mediated decrease in gene expression by TALH cells or loss and/or hypoproliferation of TALH cells, as suggested by the decreased renal medullary cell proliferation in the RAS-inhibited neonatal rats ( 21 ). Even if the downregulation of MHC II expression in TALH cells reflects merely a diminished TALH cell pool in developing AT 1 -inhibited kidneys, the upregulation of TALH-specific markers, COX-2 protein and THP, in remaining medullary TALH and cortical TALH indicates that expression of different molecules in the existing TALH cells is differentially regulated by the RAS.

Given the sensitivity of the TALH to ischemic injury ( 3, 20 ), one might speculate that a loss of MHC II expression by developing TALH cells could be secondary to a reduction in arterial pressure, and thereby decreased tissue oxygen supply, induced by losartan. However, when nifedipine, known to be a potent arterial pressure-lowering agent ( 10 ) resulting in medullary hypoxia in the neonatal pig ( 24 ), was administered to neonatal rats, the pattern of MHC II expression did not change. Moreover, adult rats treated neonatally with nifedipine exhibit normal kidney structure and urinary concentrating ability ( 10 ). On the contrary, adult rats treated neonatally with losartan develop irreversible structural abnormalities and, consequently, polyuria and polydipsia ( 6 ). Therefore, our findings suggest that hemodynamic alterations may not be the primary cause for the observed changes in TALH cells.

Our results show that neonatal AT 1 -receptor inhibition produces an increase in COX-2 protein in the cortical and medullary TALH. COX-2 is developmentally regulated ( 44 ) and strongly expressed in the neonatal rat kidney ( 33 ). It is important for nephrogenesis, given the renal dysplasia in COX-2 null mice ( 25 ). A negative feedback of ANG II on renocortical COX-2 expression was recently established in the neonatal period. AT 1 -receptor inhibition recruited cortical TALH cells to express COX-2 ( 33 ).

Thus our present results confirm the recent findings of Stubbe et al. ( 33 ), also demonstrating recruitment of cortical TALH cells to express COX-2. In addition, we demonstrate sparse COX-2 expression in medullary TALH cells from normal neonatal kidneys and induction of COX-2 in losartan-treated developing medullary TALH. If the enhancement of COX-2 expression in TALH cells from losartan-treated kidneys plays a beneficial or harmful role is not presently known, but it could be an attempt to attenuate renal abnormalities in the context of kidney development.

We observed increased expression of THP in developing TALH cells after losartan treatment. THP is thought to be linked to the function of TALH due to its exclusive production in TALH cells ( 30 ). Despite extensive studies, the function of THP remains mysterious. It is noteworthy that a modulatory role for THP in salt transport in the TALH was recently proposed. It has been suggested that THP forms a highly negatively charged, hydrophobic, gelatinous barrier in the TALH that may decrease transepithelial electrolyte flux ( 42 ). If this was the case, the AT 1 receptor inhibitor-induced anomalous overproduction of THP in developing TALH cells could hamper solute reabsorption in the TALH and lead to sodium loss, as observed in adult rats subjected to neonatal RAS inhibition ( 7, 9 ). Although our present study does not demonstrate the physiological role of THP, it suggests a causative link between RAS and THP expression during nephrogenesis.

The hypothesis that neonatal RAS inhibition may hamper the capacity of the TALH to reabsorb solutes is further supported by our finding of decreased abundance of BSC-1/NKCC2 in the developing cortical TALH, induced by losartan treatment. BSC-1/NKCC2 is expressed exclusively by TALH cells ( 5 ) and is present in renal tissue as a monomer ( 160 kDa) and/or a dimer ( 320 kDa) ( 14 ). Although we detected both (160 and 320 kDa) bands on immunoblots, only the abundance of the dimer of BSC-1/NKCC2 was significantly decreased by losartan treatment. Interestingly, a recent study demonstrated that the dimeric configuration of human BSC-1/NKCC2 exhibits significant bumetanide-sensitive Na + transport activity ( 32 ). BSC-1/NKCC2 is a primary mediator of NaCl reabsorption, taking responsibility for 25% of the body's total active NaCl reabsorption in the kidney ( 27 ). This active NaCl transport provides the energy for countercurrent multiplication, which concentrates solutes in the renal medullary interstitium, allowing generation of concentrated urine ( 12 ). Conceivably, losartan-induced decreases in the abundance of the BSC-1/NKCC2 dimer in the developing cortical TALH may contribute to the long-term impairment in urinary concentrating ability, as observed in adult rats ( 6 ).

The mechanisms underlying the differential regulation of BSC-1/NKCC2, MHC II, COX-2 and THP molecules in the developing TALH by the RAS are unclear. We conjecture that ANG II may be involved in the transcriptional regulation of BSC-1/NKCC2, MHC II, COX-2, and THP molecules. Consistent with this possibility is the finding of Stubbe at al. ( 33 ), who have demonstrated candesartan-induced renal upregulation of COX-2 at the mRNA level, and our finding of losartan-induced downregulation of MHC II mRNA in the neonatal rat kidney ( 4 ). To our knowledge, this is the first study to demonstrate the association, direct or indirect, between the RAS and BSC-1/NKCC2 and THP in the developing TALH. However, further studies are needed to determine specific molecular mechanisms implicated in losartan-mediated differential regulation of BSC-1/NKCC2, THP, MHC II, and COX-2 molecules in the developing TALH.

In summary, the main conclusion of the present study is that losartan changes the phenotype of the developing TALH, as revealed by downregulation of BSC-1/NKCC2 and MHC II and upregulation of COX-2 and THP. Collectively, our findings suggest an important role for the RAS in the development of the TALH.

GRANTS

This study was supported by the Swedish Medical Research Council (Grant 9047), the Medical Society of Gothenburg, and the John and Brit Wennerströms Research Foundation. BSC-1/NKCC2 antibody was kindly provided by Dr. Mark A. Knepper (National Heart, Lung, and Blood Institute, Bethesda, MD).

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作者单位：1 Department of Physiology, Institute of Physiology and Pharmacology, and 2 Department of Clinical Physiology, University of Gothenburg, 413 90 Gothenburg; and 3 Department of Pathology and Cytology, Karolinska Hospital, 171 76 Stockholm, Sweden