LPS decreases fatty acid oxidation and nuclear hormone receptors in the kidney^*

Materials

LPS (Escherichia coli 55:B5) was obtained from Difco and freshly diluted to the desired concentration in pyrogen-free 0.9% saline. Tri-Reagent was obtained from Sigma. Effectene transfection reagent was purchased from Qiagen. [α-³²P]dCTP (3,000 Ci/mmol) and [1-¹⁴C]oleic acid (51.0 mCi/mmol) were purchased from PerkinElmer Life Sciences. Oligo(dT)-cellulose type 77F was from Amersham Biosciences. iScript™cDNA Synthesis Kit and iQ™SYBR Green Supermix were purchased from BioRad, Hercules, CA.

Animals

Eight week-old female and male C57BL/6 mice and PPARα-deficient mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Mice were maintained in a barrier room with a normal 12 h light cycle and were provided with Harlan standard rodent chow and water ad libitum. For a typical experiment, mice were injected intraperitoneally with 100 μg of LPS in saline or with saline alone. The dose of LPS used in this study has significant effects on TG and cholesterol metabolism, but is nonlethal because the half-maximally lethal dose (LD₅₀) for LPS in rodents is ∼30–50 mg/kg body weight (3, 22). Food was withdrawn immediately after the injection of LPS because LPS induces anorexia in rodents. At the indicated time points, mice were euthanized with an overdose of halothane and kidneys were excised and stored at −80°C. All experiments were performed according to protocols approved by the Animal Studies Subcommittee of the San Francisco Veterans Affairs Medical Center.

Lipid analysis

Lipids were extracted with CHCl₃-CH₃OH-H₂O (4:2:3; by volume) from homogenized kidney lysate. Lipid composition was examined by high-performance TLC.

FA oxidation

Fresh kidneys were removed from control and 16 h LPS-treated animals and placed in cold 0.9% saline solution. Slices (0.5 mm) were then prepared with a McIlwain tissue slicer, and 100 mg portions were placed in center well 25 ml Erlenmeyer flasks. Each flask contained 2 ml Krebs-Ringer phosphate buffer (pH 7.4), 1 μCi 0.4 mM [¹⁴C]oleic acid, and 0.15 mM BSA (FA-free). Blank samples contained no tissue. Flasks were then sealed with septum caps and incubated in a shaking water bath for 2 h at 37°C. After the 2 h incubation, 0.9 ml 1 N NaOH was added to each center well through the stopper cap, and 1 ml 1 N H₂SO₄ was also added to the incubation mix through the stopper cap. Flasks were allowed to incubate an additional 15 min, after which time, flasks were removed from the water bath, caps were removed, and 450 ml aliquots of NaOH (containing trapped [¹⁴C]CO₂) were removed from center wells and added to counting vials containing 12 ml of ScintiSafe 30% LSC-counting cocktail from Fisher Scientific.

Isolation of RNA and Northern blot analysis

Total RNA from mouse kidney was isolated from 100 mg of snap-frozen tissue using Tri-Reagent. Poly(A)⁺ RNA was purified using oligo(dT) cellulose and quantified by measuring absorption at 260 nm. Ten micrograms of poly(A)⁺ RNA was denatured and electrophoresed on a 1% agarose/formaldehyde gel. The uniformity of sample loading was checked by ultraviolet visualization of the ethidium bromide-stained gel before electrotransfer to Nytran membrane (Schleicher and Schuell, Keene, NH). Prehybrization, hybridization, and washing procedures were performed as described previously (9). Membranes were probed with [α-³²P]dCTP-labeled cDNAs using Rediprime™ II random priming labeling system (Amersham, UK). mRNA levels were detected by autoradiography or by Personal FX phosphoimager (BioRad) and quantified using Quantity One software (BioRad). FACS cDNA was a gift from Dr. P. Smith (Abbott Laboratory, Columbus, OH). The mouse cDNA probes for Northern blotting were generated by reverse transcriptase PCR, starting from total RNA from mouse liver (FATP-2, ME, TRβ) and heart (TRα, CPT1α, and CPT1β) using the primers shown in Table 1.

Quantitative real-time PCR

First-strand cDNA was synthesized from 1 μg of total RNA with the iScript™cDNA Synthesis Kit (BioRad). The real-time PCR contained in a final volume 20 μl, 2 μl of cDNA, 450 nM forward and reverse primers, and 10 μl of 2 × SYBR Green PCR Master Mix (BioRad). PCR was carried out in 96-well plates using the Mx3000P™ Real-time PCR System (Stratagene, La Jolla, CA). The relative amount of all mRNAs was calculated using the comparative C_T method. 36B4 mRNA was used as the invariant control for all experiments. Quantitative PCR (QPCR) primers are listed in Table 2.

Statistical analysis

Data are expressed as the mean ± SE of experiments from four to five animals. The difference between the two experimental groups was analyzed using the Student's t-test. The difference between multiple groups was analyzed by ANOVA using the Bonferroni test.

Effect of LPS treatment on serum parameters

As seen in previous experiments, LPS administration increased serum TG (control 59.2 ± 1.4 mg/dl vs. LPS 94.4 ± 9.7 mg/dl, P < 0.01) and serum amyloid A levels (control 57.6 ± mg/dl vs. LPS 792.0 ± 4.9 mg/dl, P < 0.01). Of note, LPS treatment did not affect serum creatinine levels (control 0.77 ± 0.07 mg/dl vs. LPS 0.83 ± 0.07 mg/dl, not significant).

Effect of LPS treatment on lipid composition of kidney

Our initial experiment determined the lipid composition of mouse kidney 16 h after the administration of 100 μg LPS. As previously reported by other investigators (18), TG mass increased by 4-fold with LPS treatment (Fig. 1). Additionally, LPS treatment resulted in a 2-fold increase in total FFA levels in the kidney, whereas cholesteryl ester mass showed a moderate increase (about 30%) (Fig. 1). In contrast, phosphatidylcholine, phosphatidyl ethanolamine, and cholesterol mass were not affected by LPS treatment. These data suggest that LPS administration leads to a specific increase in FFA, TG, and cholesteryl ester levels in the kidney.

Effect of LPS treatment on FA oxidation in the kidney

When the FA [1-¹⁴C]oleic acid was used as a substrate for measuring renal FA oxidation, the production of ¹⁴CO₂ was decreased by more than 40% from kidneys obtained from LPS-treated animals, compared with controls (Fig. 2A). This indicates that FA oxidation in mouse kidney is markedly reduced by LPS treatment.

Effect of LPS treatment on genes that regulate FA metabolism in the kidney

Decreased FA oxidation following LPS treatment may result from reduced expression of several proteins required for FA oxidation. Therefore, we next determined the effect of LPS treatment on renal expression of genes that mediate FA uptake, esterification, transport, and oxidation. As shown in Fig. 2B, C, at 16 h after LPS treatment, mRNA levels of FATP-2, a dominant form of FA transporter in kidney, were decreased by 77%. However, two other FA transporters, FATP-1 and FATP-4, were not affected by LPS treatment (data not shown). LPS treatment also dramatically decreased expression of FACS, an enzyme responsible for converting FAs to fatty acyl-CoA, by 83%. Moreover, mRNA levels for two enzymes that transport FA moieties across the mitochondrial membranes, CPT1α and CPT1β, were decreased by 22% and 74%, respectively. Finally, expression of ACO and MCAD, key enzymes that mediate FA oxidation, was decreased by 36% and 68%, respectively. These data clearly indicate that the expression of a number of genes that play important roles in FA oxidation is downregulated in the kidney during the acute-phase response.

Although a decrease in the expression of any one of the aforementioned genes may diminish FA oxidation activity, it is likely that the LPS-induced impairment of FA oxidation activity is a result of a coordinate downregulation of several genes that mediate different steps along the FA oxidation pathway. In contrast to the effects of LPS on the genes involved in FA oxidation, the mRNA levels of genes involved in FA synthesis, acetyl CoA carboxylase and FA synthase, were not altered in the kidney following LPS treatment (data not shown). However, other genes regulated by PPARα in the kidney were also downregulated following LPS administration (AQP3 and Cyp4a10) (Fig. 2C).

Effect of LPS treatment on PPAR expression in the kidney

PPARα stimulates the expression of the genes in the FA oxidation pathway that were decreased in the kidney after LPS administration (11, 12). Therefore, we next determined the effect of LPS administration on the mRNA levels of the three PPAR isoforms in mouse kidney. As shown in Fig. 3A, PPARα mRNA levels were decreased by 75% in the kidney of LPS-treated animals. In contrast, the mRNA levels of PPARβ/δ and PPARγ were not altered in the kidney of LPS-treated animals. Unfortunately, due to the presence of numerous nonspecific bands, we were unable to measure PPARα protein levels in the kidney by Western blotting using several commercially available antibodies (Cayman Chemical, R and D Systems, and Santa Cruz Biotechnology). These data indicate that LPS treatment specifically decreases the expression of PPARα in the kidney. Previously, we found a 50% decrease in RXRα, which is an obligate partner for PPARα in the kidney (2, 21).

Effect of LPS treatment on the expression of TR and other nuclear hormone receptors in the kidney

In addition to PPARα, activation of TR also stimulates the expression of genes involved in FA metabolism, such as CPT1 and malic enzyme (13–15, 23). We therefore next determined the effect of LPS administration on mRNA levels of TR in the kidney 16 h after LPS treatment (Fig. 3B). We found that both TRα and TRβ mRNA levels were decreased, by 92% and 84%, respectively (similar results were seen using QPCR; TRα decreased by 75% and TRβ decreased by 71%). Consistent with the suppression of TRs, mRNA levels of malic enzyme were also decreased by 53% (Fig. 3B). In addition, we also assessed the effect of LPS treatment on the expression of other nuclear hormone receptors in the kidney. We found that the mRNA levels of HNF4 (60%), glucocorticoid receptor (54%), vitamin D receptor (83%), PXR (83%), RXRγ (78%), and ERRα (83%) all decrease with LPS treatment. In contrast, LXRβ did not change with LPS treatment.

Effect of LPS treatment on coactivators in the kidney

Recent studies by our laboratory have shown that the acute-phase response not only decreases the expression of key nuclear hormone receptors that regulate lipid metabolism but also decreases coactivators that are required for the increase in gene transcription that occurs with activation of nuclear hormone receptors (16, 17, 24). Figure 4A shows the effect of LPS treatment on coactivator mRNA levels in the kidney. Both PGC1α and -β were markedly decreased following LPS treatment (75.5% and 65.8% decrease, respectively). In contrast, mRNA levels of SRC1, SRC2, TRAP, and Lipin 1 were not affected by LPS treatment. These results demonstrate that the acute-phase response, in addition to decreasing the expression of specific nuclear hormone receptors, also decreases the expression of a subset of coactivators that regulate gene transcription.

It is well known that PGC1 regulates the expression of mitochondrial genes that play an important role in β-oxidation (25, 26). Therefore, we next determined the effect of LPS treatment on the expression of selected mitochondrial genes. As shown in Fig. 4B, the mRNA levels of Atp5g1, COX5a, Idh3a, and Ndufs8 all decreased in the kidney of LPS-treated animals.

Effect of LPS treatment on mRNA levels in males

The above studies were all carried out in female mice. Therefore, we next determined whether similar changes occur in male mice. In males, 16 h after treatment with 100 μg LPS, there was a 45% decrease in PPARα, a 68% decrease in PGC1α, a 56% decrease in PGC1β, a 64% decrease in TRα, a 54% decrease in TRβ, and a 54% decrease in FATP-2 mRNA levels in the kidney. Thus, similar decreases in kidney gene expression are induced by LPS treatment in male and female mice.

Effect of LPS treatment on mRNA levels in PPARα-deficient mice

As shown in Fig. 5, the mRNA levels of CPT1β and Cyp4a10 were markedly decreased in PPARα knock-out mice, compared with wild-type controls, whereas the levels of PGC1β, FATP2, and AQP3 were modestly decreased. In contrast, the mRNA levels of PGC1α and MCAD were similar in PPARα knock-out and wild-type mice. Following LPS treatment, the mRNA levels of PGC1α, PGC1β, CPT1β, FATP2, MCAD, AQP3, and Cyp4a10 all decreased, indicating LPS regulation of the expression of these genes independent of PPARα.