Effects of fatty acids on mitochondrial beta -oxidation enzyme gene expression in renal cell lines

Fetta Ouali, Fatima Djouadi, and Jean Bastin

Institut National de la Santé et de la Recherche Médicale U319, Université Paris VII, 75015 Paris, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Regulatory effects of fatty acids on gene expression of medium-chain acyl-CoA dehydrogenase (MCAD), a mitochondrial beta -oxidation enzyme, were investigated in rabbit kidney cell lines derived from proximal tubule (RC.SV1), thick ascending limb of Henle's loop (RC.SV2), or collecting duct (RC.SV3). Exposure to long-chain fatty acids led to significant increases (2-fold) in MCAD mRNA abundance in RC.SV1 and RC.SV2 cells; kinetics and dose-response studies established that maximal MCAD gene stimulation was reached 4 h after addition of 50 µM oleate (C18:1) in the culture medium. These effects of fatty acids were totally abolished in the presence of 1 µg/ml actinomycin D, a transcription inhibitor. Staining of cellular lipids revealed that fatty acid-induced gene stimulation could occur in the absence of cellular fatty acid accumulation. Altogether, these data indicate that small changes in cellular fatty acid flux can have direct short-term effects on fatty acid oxidation enzyme gene expression in renal cells, and this might take part in the regulation of cellular fatty acid homeostasis in response to changes in tubular fluid composition.

fatty acid metabolism; regulation; cell culture


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

FATTY ACIDS ARE ONE OF THE main energy substrates used by the kidney to supply ATP needed for ion and solute reabsorption (15). Studies run in isolated nephron segments have established a remarkable metabolic compartmentation along the renal tubule (16). The activity of mitochondrial fatty acid beta -oxidation enzymes is highest in the proximal convoluted tubule, medullary thick ascending limb, and distal convoluted tubule, i.e., in those nephron segments that exhibit the highest levels of Na-K-ATPase activity (16).

Renal energy metabolism is quite immature at birth and progressively develops during the postnatal period, in parallel with Na-K-ATPase activity (8, 10). Mitochondrial fatty acid beta -oxidation is critically important for energy production throughout the suckling period, because maternal milk is a high-fat diet containing large amounts of long- and medium-chain fatty acids. The development of fatty acid utilization in the kidney of suckling pups is reflected by the large increase in the gene expression level of medium-chain acyl-CoA dehydrogenase (MCAD), which encodes the initial enzymatic step of mitochondrial beta -oxidation (7, 9). These postnatal changes in gene expression are controlled by several hormonal factors, including the changes in glucocorticoids and thyroid hormone status that occur during the third postnatal week (7, 9). Recent data have demonstrated that the renal expression level of MCAD and other lipid metabolism genes is also controlled by dietary factors and, in particular, could vary according to the dietary fat supply. Thus switching 2-wk-old pups from maternal milk to a low-fat diet results in a coordinate downregulation of MCAD and lipid metabolism enzyme genes in the renal cortex, and this effect is rapidly reverted by restoring a high-fat diet (19). Altogether, this suggested that fatty acid-mediated gene regulation could occur in the context of postnatal development, when fatty acid supply is particularly high and there is an increasing demand for energy production through fatty acid beta -oxidation in the kidney.

Studies run in adipocytes or hepatocytes demonstrated that fatty acids can regulate the expression level of beta -oxidation enzyme genes by activating peroxisome proliferator-activated receptor-alpha (PPARalpha ), a nuclear receptor of the steroid-thyroid hormone receptor superfamily (18). Although it is established that PPARalpha is expressed to high levels in some renal epithelia (3, 14, 22), there is no evidence for a regulatory role of fatty acids in beta -oxidation enzyme genes in renal cells. The purpose of the present study was therefore to investigate possible effects of long- or medium-chain fatty acids on gene expression of MCAD, taken as a biomarker of fatty acid beta -oxidation, in renal cell lines derived from various nephron segments. The effects of fatty acids were investigated with regard to their dose-response and kinetics, over a range of physiological concentrations, and we also studied the relationship between changes in MCAD gene expression and intracellular lipid accumulation. Another purpose of this study was to assess the role of PPARalpha in mediating changes in MCAD gene expression. To address this question, the response to clofibrate, a specific PPARalpha agonist, was also studied in the renal cell lines considered.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. The cell lines used in this study, RC.SV1, RC.SV2, and RC.SV3, were the SV40-transformed renal cell lines derived from a primary culture of rabbit kidney cortical tubular cells that have been infected with the wild-type simian virus 40 (SV40) (21). These cell lines have been shown to originate from the proximal convoluted tubule (RC.SV1), from the cortical thick ascending limb (RC.SV2), or from the cortical collecting duct (RC.SV3). Cells were routinely cultured in hormonally defined medium: DMEM-Ham's F-12, 1:1, (vol/vol); 5 µg/ml transferrin; 2 mM glutamine; 30 nM sodium selenate; 5 µg/ml insulin; 5.10-8 M dexamethasone; and 20 mM HEPES, pH 7.4, as previously described (21). The cells were incubated at 37°C in a humidified atmosphere of 5% CO2-95% O2, and the medium was changed every 48 h. All experiments were performed in near-confluent 60-mm cell plates. Because the DMEM-Ham's F-12 medium contains low levels of fatty acids, cells were switched to a mixture of DMEM-Ham's F-10, which is devoid of fatty acids, 24 h before the start of the experiments. At the time of the experiments, various concentrations of fatty acid-BSA complexes (7:1) freshly prepared from 20 mM fatty acid stock solutions were added to the incubation medium, and control cells were treated with BSA alone. Some experiments were run in the presence of actinomycin D, a cellular transcription inhibitor, added to a final concentration of 1 µg/ml. RNA extractions and MCAD gene expression studies were performed after exposure of the cells to fatty acid concentrations varying from 10 to 500 µM, for periods of time varying from 2 to 24 h. We checked that raising fatty acid concentrations to 500 µM had no effects on cell viability. Clofibrate was chosen as a typical PPARalpha activator (13), and it was added to the cell culture medium at a final concentration of 1 mM for 24 h.

Northern blot analysis. Isolation of total RNA, electrophoresis through a formaldehyde-containing agarose gel (20 µg/lane for cells), and transfer to a nylon membrane followed by ultraviolet cross-linking were carried out as described elsewhere (7, 9). The membranes were probed with cDNAs labeled with [alpha -32P]dCTP using the random primer technique. For rabbit PPARalpha and MCAD, cDNA fragments of 742 and 824 bp, respectively, were obtained by RT-PCR with the use of primers previously described (6). The PCR products were purified on agarose gels and directly used for labeling. Prehybridization and hybridization were performed in a hybridization oven at 68°C, using the QuickHyb solution (Stratagene, La Jolla, CA) according to the manufacturer's instructions. The membranes were washed twice with 2× standard sodium citrate (SSC; 1x SSC is 0.15 M NaCl/0.015 M sodium citrate) for 15 min at room temperature, once with 2× SSC and 1% SDS for 15 min at room temperature, and once with 1× SSC and 1% SDS for 15-30 min at 64°C. Autoradiographs were obtained by exposing the membranes to Kodak X-OMAT film (Sigma, St. Louis, MO) with two intensifying screens at -80°C. Multiple exposures were performed to ensure that the signals were within the linear range of film sensitivity. Quantification of signal density for each mRNA was performed by computerized densitometric analysis of the autoradiograms. All the blots were finally hybridized with an 18S cDNA probe to correct for variations in the amount of total RNA loaded.

Histological studies. To reveal intracellular lipid accumulation under the various experimental conditions tested, 60-mm cell plates were stained with oil red O. After the culture medium was removed, the cells were washed three times with Hanks' balanced salt solution (HBBS; pH 7) and fixed with 10% formol for 10 min. Cells were then washed with HBBS and water and dehydrated by 100% propylene glycol. Staining was performed by adding 0.7% oil red O for 60 min at 60°C. Cells were counterstained with Harris hematoxylin to reveal nuclei.

Expression of results and statistical analysis. The mRNA levels were expressed as a relative percentage, and the results were obtained from at least two different Northern blots. The data are expressed as means ± SE. The means of four to eight cell plates in each experimental group were subjected to one-way ANOVA and Fisher's test. P < 0.05 was considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of long- and medium-chain fatty acids on MCAD gene expression in RC.SV1, RC.SV2, and RC.SV3 cells. Under standard culture conditions, MCAD mRNA abundance was highest in the RC.SV1 cells, and the levels found in the RC.SV2 and RC.SV3 accounted for 62 and 54% of this maximum value, respectively (data not shown). In the first series of experiments, MCAD gene expression was determined after exposure of the three cell lines for 24 h to either 500 µM palmitate or oleate (long-chain fatty acids C16 and C18:1, respectively) or to 500 µM octanoate or dodecanoate (medium-chain fatty acids C8 and C12, respectively). As shown in Fig. 1, medium-chain fatty acids failed to induce changes in MCAD mRNA abundance in any of the cell lines tested. Exposure to long-chain fatty acids induced significant increases in MCAD mRNA steady-state levels in RC.SV1 and RC.SV2 cells, but not in RC.SV3 cells. Palmitate had similar stimulatory effects on MCAD gene expression (+40%) in RC.SV1 and RC.SV2 cells. In contrast, oleate-induced stimulation of MCAD gene expression was higher in RC.SV1 (2.2-fold) than in RC.SV2 (+40%) cells. Cells treated with BSA only exhibited no change in gene expression compared with untreated controls (data not shown). On the basis of these results, further MCAD gene regulation experiments were performed in oleate-treated RC.SV1 cells.


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Fig. 1.   Effects of medium-chain and long-chain fatty acids on medium-chain acyl-CoA dehydrogenase (MCAD) gene expression in rabbit kidney cell lines derived from proximal tubule (RC.SV1; A), thick ascending limb of Henle's loop (RC.SV2; B), and collecting duct (RC.SV3; C) cells. The cell lines were cultured for 24 h in the presence of fatty acid-BSA complexes added to the culture medium to reach a final concentration of 500 µM octanoate (C8) or dodecanoate (C12), or palmitate (C16) or oleate (C18:1). Control cells received BSA alone. Quantification of MCAD mRNAs were then performed by Northern blot analysis; results were corrected for changes in sample loading (see METHODS). Values are means ± SE of 4-8 determinations.* P < 0.05 and *** P < 0.001 compared with control.

Dose-response and kinetics effects of oleate on MCAD gene expression in RC.SV1 cells. The effects of 10, 50, 100, or 500 µM oleate on MCAD mRNA abundance over a 24-h period were tested in RC.SV1 cells. As shown in Fig. 2, only the lowest fatty acid concentration tested (10 µM) was found to be ineffective in stimulating gene expression. Raising the oleate concentration from 10 to 50 µM induced a 1.8-fold stimulation of MCAD gene expression, and similar stimulations were observed at higher oleate concentrations (100 or 500 µM). Kinetics studies performed at 2, 4, 8, or 24 h using 500 µM oleate (Fig. 3) revealed no change in MCAD gene expression after an initial 2-h exposure to fatty acid, followed by a sharp increase in mRNA between 2 and 4 h. The mRNA induction observed after a 4-h exposure to fatty acid (2-fold) was similar to those resulting from longer (8- or 24-h) incubation periods.


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Fig. 2.   Oleate dose-response studies in RC.SV1 cells. MCAD mRNA abundance (%) was measured in RC.SV1 cells treated with various concentrations of oleate for 24 h. Control cells received BSA alone. Values are means ± SE of 4-8 determinations. *** P < 0.001 compared with control.



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Fig. 3.   Time course of oleate's effects on MCAD mRNA in RC.SV1 cells. Cells were cultured in the presence of 500 µM oleate and harvested at the different times indicated. Values are means ± SE of 4-8 determinations. *** P < 0.001 compared with 0 h.

Effects of actinomycin D on MCAD gene expression in oleate-treated RC.SV1 cells. RC.SV1 cells were incubated for 8 h in the presence of 500 µM oleate ± 1 µg/ml actinomycin D. As shown in Fig. 4, addition of oleate+actinomycin totally abolished the induction of the MCAD gene observed in cells treated by oleate only. Because actinomycin D is known to block all new transcription in the cells, these results indicate that the effect of oleate on MCAD gene expression is transcriptional.


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Fig. 4.   Effect of actinomycin D on MCAD gene induction by oleate in RC.SV1 cells. RC.SV1 cells were treated for 8 h with 500 µM oleate (+) or vehicle (-). Where indicated, 1 µg/ml actinomycin D was added to the culture medium together with oleate. Values are means ± SE of 4-5 determinations. *** P < 0.001 compared with control.

Intracellular lipid accumulation in RC.SV1 cells treated by oleate. RC.SV1 cells were stained by oil red O after a 24-h incubation with 10-500 µM oleate (Fig. 5) or after exposure to 500 µM oleate for 1-24 h (Fig. 6). Studies run at various levels of fatty acid for 24 h revealed no intracellular lipid staining in cells exposed to 10 or 50 µM oleate. Faint staining was observed beginning with exposure to 100 µM oleate, which increased with higher fatty acid concentrations. A massive intracellular lipid droplet accumulation was only observed at the highest oleate concentration tested (500 µM). Kinetics of lipid accumulation at high (500 µM) oleate concentration revealed pale microvesicular staining between 1 and 4 h after addition of oleate, which turned to a condensation of lipid droplets when cells were treated by oleate for 8 or 24 h.


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Fig. 5.   Intracellular lipid accumulation in RC.SV1 cells cultured in the presence of various oleate concentrations. Photomicrographs of RC.SV1 cells incubated for 24 h in absence of oleate (A) or in the presence of 10, 50, 100, 250, or 500 µM (B, C, D, E, and F, respectively) oleate are shown. The cells were stained with oil red O as described in METHODS.



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Fig. 6.   Intracellular lipid accumulation in RC.SV1 cells cultured in the presence of oleate. Photomicrographs of RC.SV1 cells incubated in absence of oleate (A) or in presence of 500 µM oleate for 1, 2, 4, 8, or 24 h (B, C, D, E, and F, respectively). The cells were stained with oil red O as described in METHODS.

Effects of clofibrate on MCAD gene expression in RC.SV1, RC.SV2, and RC.SV3 cells. As a first step, we determined the PPARalpha mRNA level in the three cell lines and found that it followed a distribution pattern similar to that of MCAD mRNA, i.e., highest in RC.SV1 and lower in RC.SV2 and RC.SV3 (data not shown). Figure 7 shows that exposure to clofibrate for 24 h induced changes in mRNA abundance similar to those found after treatment with oleate. Indeed, clofibrate induced significant increases in MCAD gene expression in RC.SV1 and RC.SV2 cells, but not in RC.SV3 cells.


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Fig. 7.   Effect of clofibrate on MCAD mRNA in RC.SV1 (A), RC.SV2 (B), and RC.SV3 (C) cells. MCAD mRNA abundance was measured in cells treated with 1 mM clofibrate (Clof) for 24 h. Control cells (C) received vehicle. Values are means ± SE of 5 determinations. *** P < 0.001 compared with control.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

There is growing evidence in support of a regulatory role of fatty acids in gene expression in different cell types (12, 18), but this has received little attention with regard to the kidney. The present study clearly shows that fatty acids can act as major signaling factors to regulate the expression of the MCAD gene in renal cells. A comparison of data obtained from the three cell lines demonstrates that stimulation of the MCAD gene in response to fatty acids occurred only in RC.SV1 and RC.SV2 cells, derived from the proximal convoluted tubule and cortical thick ascending limb, respectively, but not in RC.SV3 cells, which originate from the collecting duct. The mitochondrial beta -oxidation pathway is much more developed in the proximal and thick ascending limb epithelia than in the collecting duct (1, 4). Furthermore, the existence of cell-specific regulation of oxidative enzymes has been reported in the immature rat renal tubule (1, 8, 10). Our data clearly suggest that the different kidney cell lines used in the present study have maintained some degree of metabolic specificity with regard to their response to fatty acids. Our data may also suggest that beta -oxidation enzyme gene regulation only takes place in renal cells actively involved in fatty acid utilization. This is in keeping with data in the literature showing that fatty acid-mediated gene regulation operates preferentially in cell types like hepatocytes or adipocytes with extensive fatty acid metabolism capacities (12).

The regulatory effects of fatty acids on the MCAD gene were totally abolished in the presence of actinomycin D and therefore clearly result from a stimulation of gene transcription. Changes in MCAD mRNA occurred only in response to long-chain fatty acids, whereas medium-chain fatty acids were found ineffective. These data are in agreement with results from transfection experiments using constructs of MCAD gene 5'-regulatory regions together with PPARalpha expression vectors (17). In these experiments, only long-chain, but not medium-chain, fatty acids induced transactivation of the reporter gene, and this is due to the fact that medium-chain fatty acids are poor ligands of PPARalpha , as shown in a later study (13). Altogether, our data are therefore consistent with a role of PPARalpha in mediating fatty acid-induced changes in MCAD gene transcription in RC.SV1 and RC.SV2 cells. Indeed, changes in MCAD gene expression occurred shortly after exposure to fatty acids, consistent with a receptor-mediated effect. Exposure of renal cells to an agonist of PPARalpha (clofibrate) mimicked the effects of fatty acids on MCAD gene expression; in particular, MCAD stimulation in response to clofibrate occurred only in RC.SV1 and RC.SV2 cells but not in the RC.SV3 cell line. Finally, PPARalpha gene expression was found significantly higher in the RC.SV1 and RC.SV2 than in the RC.SV3 cell line (data not shown), in keeping with the distribution of PPARalpha transcripts along the rabbit kidney nephron (14). Altogether, PPARalpha appears as the best candidate to mediate fatty acid effects on gene transcription such as those observed here or in other studies (12). Definite evidence for its implication is difficult to establish, however, and is still a matter of debate (12). In this respect, it is interesting to mention that fatty acids were also reported to potentially activate the transcription factor hepatocyte nuclear factor-4 (12), which is expressed in the kidney (11) and can compete with PPARalpha in the occupancy of MCAD gene regulatory regions (5).

One of our main objectives was to study the concentration range over which fatty acids can induce changes in MCAD gene expression in renal cells. In parallel, we sought to determine whether these changes could occur in response to moderate variations in the fatty acid flux within the cells, or, rather, were triggered by massive cellular lipid accumulation. In fact, fatty acid-induced gene regulation has mainly been studied in adipocytes or liver cells (12, 18), i.e., in cell types with fatty acid storage capacities that greatly exceed those of renal cells. Furthermore, changes in gene expression reported in liver or adipose tissue were described in situations (starvation-refeeding, high-fat diet, etc.) in which large changes in intracellular lipid stores normally occur in both tissues (12). This might underline that regulation of gene expression is associated with regulated storage functions that allow large variations in the cellular pool of fatty acids. If so, it would be unlikely that these mechanisms operate in renal epithelial cells, which have limited capacities to store lipids. The data obtained in this study allow one to reach a conclusion on some of these points. Thus by comparing the effects of oleate over a physiological range of concentrations, we found that maximal stimulation of MCAD gene expression was reached for low (50 µM) oleate levels. Furthermore, cells exposed to 50 µM oleate for 24 h did not exhibit cytoplasmic lipid accumulation. It can therefore clearly be concluded that fatty acid-mediated gene regulation did not require high extracellular fatty acid concentrations, nor did it require accumulation of fatty acids within the cells. This latter point also appears from the data obtained when the cells were incubated in the presence of high (500 µM) oleate concentrations for various periods of time. In this case, maximal stimulation of MCAD gene expression was obtained 4 h after addition of oleate, at a time when lipid accumulation within the cells was yet barely detectable.

In conclusion, this study demonstrates that short-term exposure of renal cells to low extracellular levels of long-chain fatty acids can induce transcriptional changes in MCAD gene expression and that this is not related to appreciable changes in the intracellular lipid stores. The renal supply of fatty acids can vary according to changes in their plasma levels resulting from hormonal and/or nutritional factors. Regulation of fatty acid uptake by renal cells is still incompletely understood. Recent data indicate that fatty acid uptake probably involves a plasma membrane carrier protein called the fatty acid transporter, which includes six potential candidates (12). Among these, fatty acid transport protein is expressed in the rat kidney cortex, and we recently showed that fatty acid transport protein gene expression is upregulated by high-fat feeding or by administration of clofibrate (20). Whatever mechanism is involved, it is known that renal uptake of fatty acids can vary according to changes in the plasma level of these substrates (2). Our data therefore suggest that such changes in cellular fatty acid flux, even when relatively modest, could have direct short-term effects on fatty acid beta -oxidation enzyme gene expression. In renal cells, as in other cell types, these mechanisms might proceed from metabolic adaptations to physiological changes in substrate supply. Accordingly, it can be proposed that during the postnatal period, fatty acids provided by maternal milk could control the expression level of mitochondrial beta -oxidation enzyme genes in some renal cells.


    ACKNOWLEDGEMENTS

We thank Dr. P. Ronco for the gift of RC.SV cells.


    FOOTNOTES

Address for reprint requests and other correspondence: J. Bastin, INSERM U393, Hôpital Necker-Enfants Malades, 149 rue de Sèvres, 75015 Paris, France (E-mail: bastin{at}necker.fr).

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. Section 1734 solely to indicate this fact.

March 5, 2002;10.1152/ajprenal.00324.2001

Received 26 October 2001; accepted in final form 27 February 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Renal Fluid Electrolyte Physiol 283(2):F328-F334
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