Arachidonic acid metabolic pathways regulating activity of renal Na+-K+-ATPase are age dependent

Dailin Li1, Roger Belusa1, Susana Nowicki1,2, and Anita Aperia1

1 Department of Woman and Child Health, Pediatric Unit, Karolinska Institute, S-171 76 Stockholm, Sweden; and 2 Centro de Investigaciones Endocrinologicas, Consejo Nacional de Investigaciones Cíentificas y Técnicas, 1425 Buenos Aires, Argentina


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Locally formed arachidonic acid (AA) metabolites are important as modulators of many aspects of renal tubular function, including regulation of the activity of tubular Na+-K+-ATPase. Here we examined the ontogeny of the AA metabolic pathways regulating proximal convoluted tubular (PCT) Na+-K+-ATPase activity in infant and adult rats. Eicosatetraynoic acid, an inhibitor of all AA-metabolizing pathways, abolished this effect. AA inhibition of PCT Na+-K+-ATPase was blocked by the 12-lipoxygenase inhibitor baicalein in infant but not in adult rats and by the specific cytochrome P-450 fatty acid omega -hydroxylase inhibitor 17-octadecynoic acid in adult but not in infant rats. The lipoxygenase metabolite 12(S)-hydroxyeicosatetraenoic acid (HETE) and the cytochrome P-450 metabolite 20-HETE both inhibited PCT Na+-K+-ATPase in a protein kinase C-dependent manner, but the effect was significantly more pronounced in infant PCT. Lipoxygenase mRNA was only detected in infant cortex. Expression of renal isoforms of cytochrome P-450 mRNA was more prominent in adult cortex. In summary, the AA metabolic pathways that modulated the activity of rat renal proximal tubular Na+-K+-ATPase are age dependent.

renal cytochrome P-450; 12-lipoxygenase; protein kinase C; 12-hydroxyeicosatetraenoic acid; 20-hydroxyeicosatetraenoic acid


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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SEVERAL LINES OF EVIDENCE suggest that arachidonic acid (AA) metabolites play important roles in the regulation of renal function (5, 15, 27). For example, the activity of Na+-K+-ATPase in proximal tubules, collecting duct, and medullary thick ascending limb of the loop of Henle is modulated by AA metabolites (8, 18, 23, 25, 26). These effects are of great physiological importance because the enzyme Na+-K+-ATPase generates energy for the tubular absorption of Na+.

AA is released from membrane lipids in response to receptor-dependent as well as receptor-independent activation of phospholipase A2 (PLA2) (3, 6, 25). Free AA can be metabolized to biologically active metabolites by three enzyme systems, namely, the cyclooxygenase pathway, the lipoxygenase pathway, and the cytochrome P-450 monooxygenases pathway. There are indications from experimental studies that in rat kidney the cytochrome P-450 monooxygenases pathway is influenced by the state of maturation (21, 27) and may be also upregulated in pathological conditions, such as spontaneous hypertension (21, 22, 27) and salt-sensitive hypertension (14).

There are profound alterations in the regulation of Na+ excretion during ontogeny (1, 9, 20). This has prompted us to examine the ontogeny of the AA-metabolizing pathways involved in the regulation of renal tubular Na+-K+-ATPase activity. We report here that the AA-lipoxygenase pathway, that has hitherto been considered to be of little importance for the regulation of renal proximal tubular function, plays a physiological role in modulation of renal tubular Na+-K+-ATPase in infant rats. This pathway is downregulated during ontogeny. In accordance with previous studies, we found that adult rats use the cytochrome P-450 pathway to regulate proximal convoluted tubule (PCT) Na+-K+-ATPase (18, 23, 25, and this study).


    MATERIALS AND METHODS
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MATERIALS AND METHODS
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Animals. Male Sprague-Dawley rats (B&K Universal, Sollentuna, Sweden) aged 9-11 (infant) and 39-45 days (adult) were used. Body weights were 22-32 and 150-200 g, respectively. The pups were kept with their dams, and the adult rats were fed a standard rat chow (B&K Universal) ad libitum and had free access to tap water.

Chemicals. AA was purchased from Nu Chek Prep (Elysian, MN); 20- hydroxyeicosatetraenoic acid (HETE), 12(S)-HETE, and 12(R)-HETE from Cascade Biochem (Berkshire, UK); 17-octadecynoic acid (17-ODYA) from Cayman Chemical (Ann Arbor, MI); and baicalein and bisindolylmaleimide (GF-109203X) from Calbiochem (La Jolla, CA). Eicosatetraynoic acid (ETYA), nordihydroguaiaretic acid (NDGA), and all other chemicals were purchased from Sigma Chemical (St. Louis, MO). AA, 20-HETE, and 12(S)-HETE and inhibitors of AA metabolism were dissolved in ethanol. GF-109203X was dissolved in DMSO. Final concentrations of ethanol and DMSO in incubation media were shown not to interfere with Na+-K+-ATPase activity in control experiments.

Microdissection of single tubular segment and determination of Na+-K+-ATPase activity. Na+-K+-ATPase activity in PCT segments was measured as previously described (16). PCT segments (length 0.5-1 mm) were dissected from collagenase (0.05%)-perfused rat kidney. The tubular segments were preincubated with or without the addition of drugs for 30 min at room temperature in 1 µl of oxygenated modified Hanks' solution (MHS) containing (in mM) 137 NaCl, 5 KCl, 0.8 MgSO4, 0.33 Na2HPO4, 0.44 KH2PO4, 0.25 CaCl2, 1 MgCl2, and 10 Tris · HCl. Butyrate (10-3 M) was added to the solution to optimize mitochondrial respiration. After preincubation in MHS with or without drugs, MHS was removed and ice-cold ATPase assay solution, with compositions described below, was added. The segments were permeabilized with rapid freezing and thawing in the ATPase assay solution to ensure that Na+ and ATP entered the cell. The Na+ concentration in the medium was 70 mM. Permeabilization clamps the intracellular Na+ concentration and the Na+ concentration in the medium, thereby eliminating the transmembrane Na+ gradient and the possibility that changes in Na+-K+-ATPase activity are secondary to changes in intracellular Na+ concentration. All Na+-K+-ATPase assays were performed in the presence of saturating concentrations of all major substrates (70 mM Na+, 5 mM K+, and 10 mM ATP). After preincubation and permeabilization, the tubule segments were incubated for 15 min at 37°C in the ATPase assay solution (in mM): 50 NaCl, 5 KCl, 10 MgCl2, 1 EGTA, 100 Tris · HCl, and 10 Na2ATP, as well as 2-5 Ci/mmol [gamma -32P]ATP in tracer amounts (5 nCi/µl), pH 7.4, at 37°C with or without 2 mM ouabain. When ouabain was present, NaCl and KCl were omitted and Tris · HCl was 150 mM. The phosphate liberated by hydrolysis of [gamma -32P]ATP was separated by filtration through a Millipore filter after absorption of the unhydrolyzed ATP on activated charcoal. The radioactivity was measured in a liquid scintillation spectrophotometer. In each study (each rat), total ATPase activity was measured in five to eight segments, and ouabain-insensitive ATPase activity was measured in five to eight segments. Na+-K+-ATPase activity was calculated as the difference between the means of the total ATPase and the ouabain-insensitive ATPase activity and was expressed as picomoles 32P hydrolyzed per millimeter tubule per hour. Results are given as the percentage of control.

Purification of rat renal Na+-K+-ATPase. Na+-K+-ATPase was purified from adult rat renal cortex as described (11). The preparation was analyzed by SDS-PAGE. The gel was stained with Coomassie brilliant blue. Densitometric analysis of the stained gel indicated that the Na+-K+-ATPase alpha -subunit constituted ~55% of total proteins in the preparation. The specific activity of Na+-K+-ATPase in the preparation was calculated as ~700 µmol of ATP hydrolyzed · mg protein-1 · h-1. The ouabain (5 mM)-insensitive ATPase activity was <3% of total ATPase activity.

Phosphorylation of purified Na+-K+-ATPase by protein kinase C (PKC). Assays were carried out at 24°C in a reaction volume of 20 µl containing (in mM) 50 HEPES (pH 7.5), 0.85 CaCl2, 10 MgCl2, and 1 EGTA, as well as 10 nM PKC and 1 µg of purified Na+-K+-ATPase. Reactions were initiated by the addition of ATP (final concentration 0.1 mM), with a trace of [gamma -32P]ATP (2,000-3,000 cpm/pmol). Reactions were stopped by the addition of Laemmli sample buffer. Samples were resolved by 7.5% SDS-PAGE. Gels were stained with Coomassie brilliant blue, destained, dried, and analyzed by autoradiography or the Bio-Rad Molecular Imager Phosphor Imaging System (model GS-250).

Gene expression analysis by semiquantitative RT-PCR. Renal cortical slices (200 µm thick) from the superficial cortex were used. These slices consist mainly (>90%) of proximal tubules (2). Because leukocytes and thrombocytes express lipoxygenase (34), blood was removed from the renal vasculature by perfusing the left kidney with cold Ringer solution until blanched. Quantitative microscopy inspection of digested slices showed that fewer than two glomeruli per slice were present in slices from either adult or infant rats (data not shown).

The method for RT-PCR was modified from the one previously described (36). Reverse transcription was carried out in a 10-µl reaction volume containing 1 µg total RNA, 25 mM Tris · HCl (pH 8.3), 37.5 mM KCl, 1 mM deoxynucleotide triphosphates (dNTPs), 5.0 mM dithiothreitol (DTT), 0.4 µM oligo(dT15) primer, 20 U RNasin, 80 U Moloney murine leukemia virus RT, and RNase H Minus (Promega) and incubated for 60 min at 42°C, followed by 10 min at 95°C. The RT reaction was converted to a 100-µl PCR containing 2.5 U thermostable DNA polymerase (AmpliTaq Gold, Stratagene), 0.2 µM of each gene-specific primer (see Table 1), 200 µM dNTPs, 300 nCi [32P]dCTP, 10 mM Tris · HCl (pH 8.8), 1.5 mM MgCl2, 50 mM KCl, and 0.1% Triton X-100 and sequentially cycled for 20-35 cycles. The reaction mixture was incubated in a Perkin-Elmer Cetus 9600 thermocycler (55°C for 45 s, 72°C for 90 s, 95°C for 30 s), starting at 95°C for 10 min and finishing at 72°C for 5 min. All gene-specific primer pairs used in the RT-PCR protocols are shown in Table 1. beta -Actin was used as an internal standard. Twenty microliters of the PCR product solution were separated on a 4% nondenaturating polyacrylamide gel. All the different RT-PCR products were subjected to restriction enzyme analysis and sequencing (PRISM Ready Reaction DyDeoxy Terminator Cycle sequencing kit, Applied Biosystems) to verify product specificity (data not shown). No products appeared if RT was omitted from the RT reaction solution, confirming that the products were produced from cDNA and not contaminating genomic DNA. For each variable studied, at least three different RNA preparations were analyzed, and each preparation was analyzed in triplicate. DNA molecular weight marker VI (Boehringer Mannheim) was used on all gels. The polyacrylamide gel was placed on Whatman 3 MM filter paper and wrapped in plastic. The gels were exposed to a Phosphor Imaging Screen Cassette-BI and the Bio-Rad Molecular Imager Phosphor Imaging System (model GS-250) was used to detect incorporation of radioactive dCTP. All quantitative measurements were made within linear amplification range.

                              
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Table 1.   Specific primers used in RT-PCR

To determine the efficiency of the primers in the RT-PCR protocols, total RNA from unperfused whole kidney was used. With this preparation, all primer pairs resulted in high amount of products after 30 cycles of PCR.

Statistical analysis. Values represent means ± SE. Data were analyzed by the Student's t-test or ANOVA-test. P values < 0.05 are considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Under basal condition, Na+-K+-ATPase activity (in pmol Pi · mm-1 · h-1) was 909 ± 34 (n = 20) in infant rat PCT and 1,663 ± 147 (n = 27) in adult rat PCT. AA produced a dose-dependent inhibition of the activity of Na+-K+-ATPase in PCT from both infant and adult rats. The maximal inhibition was ~45% (Fig. 1A). The concentrations required to evoke PCT Na+-K+-ATPase inhibition were somewhat lower in infant rats. The inhibitory effect of AA was completely abolished by a specific PKC inhibitor, GF-109203X (1 µM), in both infant and adult rats (Fig. 1B). Basal Na+-K+-ATPase activity was not altered by GF-109203X alone (adult rats: control 2,023 ± 190, GF-109203X 2,130 ± 268; infant rats: control 974 ± 45, GF-109203X 920 ± 80 pmol Pi · mm-1 · h-1).



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Fig. 1.   A: concentration-dependent inhibition of Na+-K+-ATPase activity by arachidonic acid (AA) in proximal convoluted tubule (PCT) from infant and adult rats. Individual tubular segments were incubated at room temperature for 30 min in the absence (-) or presence of (+) indicated concentrations of AA. Each point represents means ± SE of 4-5 separate experiments. P < 0.05 by ANOVA. B: effect of protein kinase C (PKC) inhibitor on AA inhibition of Na+-K+-ATPase activity. Individual tubular segments were incubated at room temperature for 30 min in the absence or presence of maximal concentration of AA (10 nM and 1 µM for infant and adult rats, respectively). GF-109203X (1 µM) was present 5 min before and throughout incubation with AA. Each bar represents means ± SE of 3-4 separate experiments. Statistical comparison among groups was done by ANOVA followed by Tukey-Kramer test. * P < 0.05 vs. control.

To assess whether the inhibitory effect of AA was mediated by its metabolites, inhibitors of different AA metabolic pathways were used. ETYA is an inhibitor of all AA enzymatic metabolizing pathways (26, 32). Figure 2 shows that ETYA completely blocked AA-induced Na+-K+-ATPase inhibition in PCT from both infant and adult rats. This observation rules out a direct effect of AA and strongly suggests the involvement of AA metabolites in the inhibition of renal tubular Na+-K+-ATPase. The major AA metabolic pathway operating in the proximal tubules from adult rats involves the cytochrome P-450 AA omega -hydroxylase pathway, resulting in the generation of 20-HETE (22, 27). Alternatively, AA may be also oxygenated by 12-lipoxygenase present, for instance, in platelets and vascular smooth muscle cells (17), leading to the formation of 12(S)-HETE. A specific inhibitor of the cytochrome P-450 pathway, 17-ODYA (1 µM) (37), completely prevented the effect of AA in adult but had no effect in infant rats. Two inhibitors of the lipoxygenase pathway were used, namely, 5 µM NDGA and 5 µM baicalein (30, 31). At the present time baicalein is considered to be the most specific inhibitor of 12-lipoxygenase. Both NDGA and baicalein blunted AA-induced Na+-K+-ATPase inhibition in infant but had no effect in adult rats (Fig. 2). The inhibitors, added singly, had no effect on Na+-K+-ATPase activity (adult rats: control 2,159 ± 185, 5 µM ETYA 1,945 ± 333, 1 µM 17-ODYA 2,038 ± 234, 5 µM NDGA 2,475 ± 79, 5 µM baicalein 2,084 ± 260; infant rats: control 868 ± 103, 10 nM ETYA 883 ± 85, 1 µM 17-ODYA 1,054 ± 208, 5 µM NDGA 928 ± 161, 5 µM baicalein 761 ± 42 pmol Pi · mm-1 · h-1).




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Fig. 2.   A: schematic representation of AA-metabolizing pathways, showing inhibitors of major pathways and final products investigated in present study. B: effect of inhibitors on Na+-K+-ATPase activity in PCT from infant rats. Individual tubular segments were incubated in absence or presence of maximal concentration of AA (10 nM). Inhibitors were present 5-10 min before and throughout incubation with AA. Each bar represents means ± SE of 3-4 separate experiments. Statistical comparison among groups was done by ANOVA followed by Tukey-Kramer test. * P < 0.05 vs. control. C: effect of inhibitors on Na+-K+-ATPase activity in PCT from adult rats. Individual tubular segments were incubated in absence or presence of maximal concentration of AA (1 µM). Inhibitors were present 5-10 min before and throughout incubation with AA. Each bar represents means ± SE of 3 separate experiments. BACL, baicalein; 17-ODYA, 17-octadecynoic acid; ETYA, eicosatetraynoic acid; HETE, hydroxyeicosatetraenoic acid; NDGA, nordihydroguaiaretic acid. Statistical comparison among groups was done by ANOVA followed by Tukey-Kramer test. * P < 0.05 vs. control.

We next tested the effect of metabolites from the lipoxygenase and the cytochrome P-450 pathways, 12(S)-HETE and 20-HETE (Fig. 3). In this protocol, control values in infant rats were 1,046 ± 99 (n = 6) and 858 ± 117 (n = 6) for 12(S)-HETE and 20-HETE groups, respectively; in adult rats they were 2,577 ± 35 (n = 6) and 2,850 ± 112 pmol Pi · mm-1 · h-1 (n = 10) for 12(S)-HETE and 20-HETE groups, respectively. The lipoxygenase metabolite 12(S)-HETE inhibited Na+-K+-ATPase in both infant and adult PCT, but inhibition was significantly (P < 0.01) more pronounced in the infant rats. In contrast, the cytochrome P-450 metabolite 20-HETE inhibited Na+-K+-ATPase activity to a similar extent in infant and adult PCT. Inhibition by 12(S)-HETE and 20-HETE was abolished by the specific PKC inhibitor GF-109203X in both infant and adult PCT (data not shown). 12(R)-HETE, another cytochrome P-450 metabolite that has been shown to inhibit PCT Na+-K+-ATPase in adult rats (23), was also found to inhibit the Na+-K+-ATPase in infant rats (data not shown).


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Fig. 3.   Inhibition of Na+-K+-ATPase activity by 12(S)- and 20- HETE in proximal tubules from infant and adult rats. Individual tubule was incubated for 30 min at room temperature in absence (control) or presence of 1 µM of 12(S)- or 20-HETE. 12(S)-HETE (0.1 µM) also inhibited Na+-K+-ATPase activity in infant rats, comparable to effect of 1 µM of 12(S)-HETE (data not shown). Statistical comparison among groups was done by 2-way ANOVA. * P < 0.01.

20-HETE has been recently shown to contribute to the activation of PKC (18). 12(S)-HETE was shown here to have a similar effect. In vitro PKC-dependent phosphorylation of the purified rat renal Na+-K+-ATPase alpha -subunit was increased in the presence of 12(S)-HETE at the physiological Ca2+ concentration of 1.3 µM (Fig. 4).


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Fig. 4.   Effect of 12(S)-HETE on PKC-dependent Na+-K+-ATPase phosphorylation. Purified rat renal Na+-K+-ATPase (1 µg) was incubated at 24°C for 20 min with 10 nM PKC in absence (lane 1) or presence (lane 2) of 20 µM 12(S)-HETE. Samples were subjected to SDS-PAGE. 32P-labeled proteins were analyzed by autoradiography. Bottom band in each lane shows autophosphorylation of PKC. Similar results were seen in 4 separate experiments.

The mRNA expression of 5-lipoxygenase, 12-lipoxygenase, and the cytochrome P-450 4A1 and 4A2 isoforms was semiquantitatively evaluated by use of RT-PCR. No signal from 5-lipoxygenase mRNA was detected in slices from outer cortex of infant and adult kidneys (Fig. 5), which confirmed that the slices were indeed deprived of blood and glomeruli-free (see Gene expression analysis by semiquantitative RT-PCR). 12-Lipoxygenase mRNA signal was only detected in cortical tissue from infant rats (Fig. 5). The samples were run at 25, 30, and 35 cycles. Shown in Fig. 5 are samples run at 35 cycles. 12-Lipoxygenase mRNA was not detected even at 35 cycles in samples from adult rats. Cytochrome P-450 4A1 mRNA signal was detected in both infant and adult rats at approximately equal levels (Fig. 6A). However, there were pronounced qualitative and quantitative differences between infant and adult rats with regard to the mRNA signal of cytochrome P-450 4A2 isoform. Two alternative spliced forms of the cytochrome P-450 4A2 isoform were observed and confirmed with sequencing in cortex from adult rat. The longer form had 389 bp, and the shorter form had 267 bp. The splicing occurs in exon 12. The longer transcript was expressed in both age groups, but it was 3.6-fold more abundant in adult than in infant rats (Fig. 6, B and C). The shorter transcript was strongly expressed in adult rats but was not detectable in infant rats (Fig. 6, B and C). The total level of the two transcripts for cytochrome P-450 4A2 was ~33-fold higher in adult than in infant rats (Fig. 6C).


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Fig. 5.   RT-PCR amplification of 5- (5-LO) and 12-lipoxygenase (12-LO) in renal outer cortical slices from infant and adult rats. 12-LO-RT, negative control of 12-lipoxygenase, where RT was omitted from reaction medium; BACT, beta -actin; M, molecular weight markers. beta -Actin was used as control of RNA quality. Lanes 1-4, infant rats; lanes 5-7, adult rats.



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Fig. 6.   A: RT-PCR amplification of cytochrome P-450 4A1 in renal outer cortical slices from infant and adult rats. -RT, negative control of cytochrome P-450 4A1, where RT was omitted from reaction medium. PCR was carried out for 35 cycles. Signal from cytochrome P-450 mRNA was also detected when PCR was carried out for 30 cycles (data not shown). CYP450 4A1, cytochrome P-450 4A1 isoform; I, infant rats; A, adult rats. B: RT-PCR amplification of cytochrome P-450 4A2 in renal outer cortical slices from infant and adult rats. If RT was omitted from reaction medium, no products were detected. CYP450 4A2, cytochrome P-450 4A2 isoform. Top, no. of PCR cycles. C: semiquantitative analysis of cytochrome P-450 4A2 gene expression as in B (n = 3/group). Value are means ± SE. Value was calculated as ratio of mRNA signals of cytochrome P-450 4A2 to those of beta -actin. * P < 0.05 vs. infant group by Student's t-test.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
REFERENCES

The PLA2-AA signaling pathway has recently received a great deal of attention because of its role in the regulation of Na+-K+-ATPase activity (18, 23, 25, 26) and of other tubular transporters. The first messengers activating renal PLA2 include dopamine, vasopressin, bradykinin, parathyroid hormone, and angiotensin (23, 25, 26). Only the cytochrome P-450 metabolites are responsible for the regulation of Na+-K+-ATPase in PCT from adult rats (23, 25). The cytochrome P-450-AA-omega -hydroxylase pathway is the predominant AA metabolic pathway for the regulation of PCT Na+-K+-ATPase in adult rats (13, 22, 27). The present results indicate that, in infant rat renal PCT, the lipoxygenase pathway plays the predominant role for the regulation of Na+-K+-ATPase activity and that this pathway is downregulated during postnatal maturation of the kidney. Thus 12-lipoxygenase mRNA was expressed in PCT cells from infant rats but not detectable in adult rats. The AA-lipoxygenase pathway was involved in the inhibition of PCT Na+-K+-ATPase in infant rats but not in adult rats. To our knowledge, this is the first demonstration of a physiologically active AA lipoxygenase metabolite in renal PCT. It is well established that lipoxygenase metabolites regulate cellular responses in inflammation and immunity (24). In addition, they may also have growth-promoting effects. 12(S)-HETE mediates cytokine- and phenylephrine-induced mitogenesis in vascular smooth muscle cells (17). It is therefore possible that in the infant kidney 12(S)-HETE may also be an important factor in the regulation of the growth and maturation of nephrons.

Schwartzman and co-workers (29) were the first to demonstrate that cytochrome P-450 metabolites of AA could inhibit the activity of Na+-K+-ATPase. The same group has also reported that the formation of 20-HETE remains at comparable levels from the fetal stage to 3 wk of age in Wistar-Kyoto kidney cortex. From 3 to 5 wk of age there is an abrupt increase in cytochrome P-450-AA-omega -hydroxylase activity (responsible for generation of 20-HETE), which lasts until 13 wk of age (21). These functional changes can well be explained by increased mRNA expression of cytochrome P-450 isoforms. The cytochrome P-450 4A gene family, responsible for AA omega -hydroxylation, comprises several isoforms with great homology (28). Here we have studied the cytochrome P-450 4A1, which has been considered to be the predominant isoform catalyzing the formation of 20-HETE from AA in the rat PCT (13) and the cytochrome P-450 4A2 isoform because it is hormonally regulated (10) and therefore may be developmentally regulated. We observed that although there was no detectable difference between the expression of the cytochrome P-450 4A1 mRNA in infant and adult kidney, there were pronounced qualitative and quantitative differences between infant and adult rats with regard to the mRNA signal of the cytochrome P-450 4A2 isoform. The data on the effects of the AA metabolites on Na+-K+-ATPase activity suggested that the cytochrome P-450 pathway participates in the inhibition of PCT Na+-K+-ATPase in adult rats but is of minor physiological importance in infant rats. Taken together, our results indicate that the AA-lipoxygenase pathway is downregulated and the AA-cytochrome P-450 pathway may be upregulated with age.

It has been shown that cytochrome P-450 4A1 mRNA exhibits alternative splicing (7). We observed splicing of the cytochrome P-450 4A1 mRNA in both infant and adult kidney. The splicing of cytochrome P-450 4A2 mRNA is a novel finding. The splicing of cytochrome P-450 4A2 mRNA only occurred in adult kidney, suggesting that it is developmentally regulated. In both cytochrome P-450 4A1 and 4A2 isoforms, splicing occurs in downstream, untranslated regions. This alternative splicing may be relevant to tissue- and age-specific regulation of the expression of the gene (Ref. 7 and this study).

GF-109203X, which is a highly specific PKC inhibitor (33), abolished the effects of AA, 20-HETE, and 12(S)-HETE on the activity of PCT Na+-K+-ATPase. The lipoxygenase metabolite 12(S)-HETE enhanced PKC-induced phosphorylation of Na+-K+-ATPase at physiological Ca2+ concentrations. Taken together, these data indicate that lipoxygenase products of AA inhibit PCT Na+-K+-ATPase via PKC activation.

In conclusion, the AA metabolic pathways modulating renal tubular Na+-K+-ATPase are developmentally regulated. We present here both physiology and molecular biology data that suggest that the pathways shift from a lipoxygenase-dependent pathway in infant kidney to a cytochrome P-450-dependent pathway in adult kidney. If this shift in the signaling pathways during ontogeny turns out to be a principle that is present in many tissues, the finding would be of general interest for the understanding of postnatal maturation. It will also be important to find out the physiological significance of present finding for regulation of sodium excretion.


    ACKNOWLEDGEMENTS

We thank Lill-Britt Svensson and Dr. Ann-Christine Eklöf for experimental assistance.


    FOOTNOTES

This study was supported by grants from the Swedish Foundation for International Cooperation in Research and Higher Education (to A. Aperia and S. Nowicki), the Swedish Medical Research Council, and the Märta and Gunnar V. Philipson Foundation (to A. Aperia).

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

Address for reprint requests and other correspondence: A. Aperia, Dept. of Woman and Child Health, Pediatric Unit, Astrid Lindgren Children's Hospital, Karolinska Hospital, S-171 76 Stockholm, Sweden (E-mail: aniap{at}child.ks.se).

Received 17 May 1999; accepted in final form 30 November 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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