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作者：Yahua Zhang,, Xiaoyan Zhang,, Lihong Chen, Jing Wu, Dongming Su, Wendell J. Lu, Mei-Tsuey Hwang, Guangrui Yang, Shuo Li, Minfen Wei, Linda Davis, Matthew D. Breyer, and Youfei Guan,作者单位：1 Division of Nephrology, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee; and 2 Peking University Diabetes Center, Department of Physiology and Pathophysiology, Peking University Health Science Center, Beijing, China
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【摘要】, w* A) }& {( `( D! r1 N
Liver X receptors (LXRs), including LXR and LXR, are intracellular sterol sensors that regulate expression of genes controlling fatty acid and cholesterol absorption, excretion, catabolism, and cellular efflux. Because the kidney plays an important role in lipid metabolism and dyslipidemia accelerates renal damage, we investigated the effect of TO-901317, an LXR agonist, on the gene expression profile in mouse kidney. Treatment of C57 Bl/6 mice with TO-901317 (3 mg·kg -1 ·day -1 ) for 3 days resulted in 51 transcripts that were significantly regulated in the kidney. Among them, the stearoyl-CoA desaturase-1 (SCD1) was upregulated most dramatically. Northern blot analysis revealed that SCD1 mRNA levels were markedly higher than that in control kidneys. Enhanced SCD1 expression by TO-901317 also resulted in increased fatty acid desaturation in the kidney. In control mice, constitutive renal SCD1 expression was low; however, TO-901317 treatment markedly increased SCD1 expression in the outer stripe of the outer medulla as assessed by both in situ hybridization and immunostain. Double-labeling studies further indicated that SCD1 mRNA was selectively expressed in proximal straight tubules negative for aquaporin-2 and Tamm-Horsfall protein. In vitro studies in cultured murine proximal tubule cells further demonstrated that LXR activation enhanced SCD1 transcription via increased sterol regulatory element binding protein-1. Taken together, these data suggest LXR activation of SCD1 expression may play an important role in regulating lipid metabolism and cell function in renal proximal straight tubules. ) A6 O( h8 h+ ?! J+ P) }( Y
【关键词】 gene expression stearoylcoenzyme A desaturase sterol regulatory element binding protein lipid metabolism
( m+ F! m$ X( D5 D. d3 D4 q+ I THE LIVER X RECEPTORS (LXRs) are members of the superfamily of nuclear hormone receptor of ligand-activated transcription factors that were originally identified in the liver. Two isoforms of LXRs exist, designated LXR (NR1H3) and LXR (NR1H2). They share high sequence homology and similar protein structure ( 9, 17 ). Both LXR and LXR bind as heterodimers with retinoic acid receptor RXR- to a specific DNA sequence named LXR response element in the promoter regions of their target genes, thereby modulating the target gene transcription. Target genes of LXRs include cytochrome P -450 7A1 (CYP7A1), ATP-binding cassette transporter (ABC) A1, ABCG5, ABCG8, apolipoprotein E, cholesterol ester transport protein, lipoprotein lipase, fatty acid synthase, and sterol response element-binding protein 1c (SREBP1c), consistent with a key role for LXRs in regulation of cholesterol and fatty acid metabolism ( 34 ). The LXR ligand TO-901317 significantly improves insulin resistance and lowers plasma glucose level by inhibition of hepatic gluconeogenesis in type II diabetic rats ( 6 ). Recent studies demonstrate that an LXR-specific activator increases high-density lipoprotein (HDL) cholesterol in mice, whereas gene disruption of LXR and LXR increases low-density lipoprotein cholesterol and decreases HDL cholesterol in plasma. LXR gene knockout is also associated with massive lipid accumulation in macrophages in the liver, spleen, and lung ( 26, 32 ). These findings suggest that LXR plays an important role not only in cholesterol and fatty acid metabolism but also in insulin sensitivity and glucose homeostasis.4 f; ^2 \! w3 E3 l0 ^
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Although LXR and LXR display distinct tissue distribution patterns, both are expressed in the kidney ( 3, 17, 37 ). A recent report shows that LXR mRNA is ubiquitously expressed along the nephron, and its activation mediates cholesterol efflux via ABCA1 in cultured glomerular mesangial cells ( 37 ). LXR is also widely expressed at high levels in the late stage of embryonic kidney ( 3 ). The role of these receptors in the kidney with respect to lipid metabolism is largely unknown. To provide insight into the role of LXRs in renal physiology, the present study utilized Affymetrix GeneChip to perform a systematic study of the genomewide expression profiles of the kidney from mice treated with an LXR-specific agonist, TO-901317. We provide evidence suggesting that treatment of the LXR agonist TO-901317 markedly increases the gene expression of stearoyl-CoA desaturase-1 (SCD1), possibly via increased SREBP1 expression in the proximal straight tubules. The results suggest that LXR may play an important role in regulating lipid metabolism and maintaining proximal tubule function., }3 f3 Y, d( I1 N% f; j9 D
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MATERIALS AND METHODS
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Chemical reagents. TO-901317 was obtained from Cayman Chemical (Ann Arbor, MI). Goat anti-mouse SCD1 antibody, rabbit anti-human SREBP1c antibody, and goat anti-human Tamm-Horsfall protein antibody were purchased from Santa Cruz (Santa Cruz, CA) and Organon-Technika.3 I) ]# W [/ w2 R6 s
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Animal treatment. Male C57 Bl/6 mice (8 wk old) were purchased from the Jackson Laboratories (Bar Harbor, ME) and fed with mouse chow ad libitum. At 10 wk of age, animals were treated with LXR agonist TO-901317 (3 mg·kg -1 ·day -1 ) or vehicle alone by gavage for 3 days. At the end of study, mice were killed, and tissue samples were snap-frozen in liquid nitrogen and then stored at -80°C until further processes. Kidney samples were also fixed in 4% paraformaldehyde solution for immunostaining and in situ hybridization.* \4 @2 g+ K( O9 B) k6 H0 d' ?3 `
$ l/ d+ d9 k& z# G# _RNA extraction and purification. Total RNA from murine kidneys and cultured renal cells was isolated by using Trizol-Reagent (GIBCO-BRL). The tissue and cell samples were homogenized in 10 ml Trizol reagent. Phase separation of RNA was performed by adding one-tenth volume of chloroform and vortex mixing for 15 s followed by centrifugation at 12,000 g for 10 min. Isopropyl alcohol (0.5 ml/1 ml Trizol) was added to the aqueous phase to precipitate total RNA, followed by two washes with 75% ethanol. For Affymetrix analysis, the RNA sample was dried and then redissolved. RNA quality was determined by the ratio of absorbance at 260 nm to that at 280 nm (A260/A280). Total RNA extracted was further purified using the RNeasy Clean Up kit (Qiagen) to increase the A260/A280 readings.! R3 N/ i/ L; D: g, F5 g# U
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Affymetrix GeneChip analysis. Total RNA isolated from three controls and three treated kidneys using Trizol reagent described above was treated with RNeasy to obtain a ratio of optical density at 260 to 280 nm between 1.9 and 2.1 for each sample. Gene expression level of each sample was studied using the Affymetrix mouse chip at the Vanderbilt Microarray Shared Resource (VMSR; http://array.mc.vanderbilt.edu/products/affymetrix.htm ). The chip measured the expression of 12,488 probe sets. The Signal Log Ratio (SLR) estimates the magnitude and direction of change of a transcript when two arrays are compared (experiment vs. control). A transcript is significantly increased when the SLR 1.0, indicating a twofold increase, and significantly decreased when the SLR is less than -1.0, indicating a twofold decrease. The gene expression data were analyzed using Affymetrix Microarray Suite version 5.0 and Gene Traffic at VMSR. The data were further analyzed independently using Gene Spring.
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4 T) g% n2 |7 t: h t' | QLipid analysis. Fatty acid profiles of lipid esters in the kidneys were analyzed by gas-liquid chromatography at Vanderbilt Lipids/Lipid Peroxidation Core. Briefly, total lipids were extracted from mouse kidneys and were separated by silica gel TLC using hexane-diethyl ether-acetic acid (80:30:1 vol/vol/vol) as a developing solvent. The lipids were visualized by cupric sulfate in 8% phosphoric acid. The lipids were then scraped, methylated, and analyzed by gas-liquid chromatography ( 20 )." ~5 G. P, T) t0 E( d* H4 q
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RT-PCR analysis. To determine whether the LXRs are expressed in mouse kidney and cultured murine proximal tubule cells (MCT cells; see Ref. 14 ), RT-PCR analysis was utilized. Total RNA isolated from three normal male C57Bl/6 mice (10 wk old) was reverse transcribed to single-stranded cDNA using Moloney murine leukemia virus RT and 2.5 µM of random hexamers according to the manufacturer's protocol (GeneAmp RNA PCR kit; Perkin-Elmer Cetus, Norwalk, CT). The cDNAs were then amplified using LXR, LXR, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) selective primers. Mouse LXR primer sequences are 5'-TCC ATC AAC CAC CCC CAC GAC-3' for sense and 5'-CAG CCA GAA AAC ACC CAA CCT-3' for antisense. Mouse LXR primer sequences are 5'-TCG CCA TCA ACA TCT TCT CAG for sense and 5'-GTG TGG TAG GCT GAG GTG TAA-3' for antisense. Mouse GAPDH primer sequences are 5'-TCC GTG TTC CTA CCC CCA ATG-3' for sense and 5'-GAG TGG GAG TTG CTG TTG AAG-3' for antisense. These primers were used to amplify a 328-bp product from LXR, a 514-bp product from LXR, and a 178-bp GAPDH cDNA fragment. PCR reactions were carried out in 10 mM Tris·HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl 2, 0.2 mM dNTPs, and 1 µM primers at 94°C for 0.5 min, 60°C for 0.5 min, and 72°C for 1.0 min for 35 cycles in a Perkin-Elmer Cetus 2400 thermal cycler. PCR products were separated by 1% agarose gel and further confirmed by sequencing.5 e" Z; w: s$ B5 G( y
$ B0 u) U3 i3 g& E, Z, wRT-PCR was also used to amplify a 352-bp product of SCD-1 cDNA fragment from mouse fat tissues for use in in situ hybridization. A pair of primers with the sequences 5'-CTT CTT GCG ATA CAC TCT GGT-3' for sense and 5'-AGG AAC TCA GAA GCC CAA AGC-3' for antisense were used. The resulting 352-bp PCR fragment was cloned into pCRII2.0 vector (Invitrogen) and sequenced.' M* s; `" p+ t5 J0 \* {; @. V
. N2 b4 f. C$ b9 v0 DNorthern blot analysis. Total RNA was extracted from the kidneys of mice treated with or without the LXR agonist TO-901317. Total RNA (20 µg) was size-separated in 1% agarose gel and transblotted to a nylon membrane; the blot was prehybridized in 6 x saline-sodium citrate (SSC), 5 x Denhardt's solution, 1% SDS, and 150 µg/ml of freshly denatured salmon sperm DNA for 4 h and then hybridized in same buffer with 1 x 10 6 counts·min -1 ·ml -1 of 32 P-labeled mouse SCD1 or SCD2 cDNA probe (a gift from Dr. N. Wang at the Institute of Cardiovascular Science, Peking University Health Science Center) at 62°C overnight. After hybridization, the blot was washed one time in 2 x SSC, 0.1% SDS for 30 min at room temperature followed by two washes in 0.1 x SSC, 0.1% SDS at 62°C for 30 min and exposed to X-ray film at -70°C for 40 min.4 f4 B4 c: j2 u" y
) B' _+ k2 b- p3 y8 j+ Q/ YIn situ hybridization. In situ hybridization was performed as previously reported ( 10 ). Riboprobes were made for in situ hybridization using the 352-bp product from mouse SCD1 cDNA. The plasmid containing mouse SCD1 cDNA was linearized, and 35 S-UTP-labeled antisense riboprobe was synthesized and hybridized to mouse kidney sections and then washed as previously described ( 10 ). Slides were dehydrated with graded ethanol containing 300 mM ammonium acetate, dipped in emulsion (Ilford K5; Knutsford, Cheshire, UK), and exposed for 4-5 days at 4°C. After being developed in Kodak D-19, some slides were counterstained with hematoxylin, and photomicrographs were taken using a Zeiss Axioskop microscope and either darkfield or brightfield optics.3 C6 U. s; |0 X3 m8 g
n0 @ W4 [: a& T2 G; F1 D) m* uImmunohistochemistry. Immunohistological examination and additional staining of in situ hybridization slides was performed using a goat anti-mouse SCD1 antibody (1:500; Santa Cruz), rabbit anti-aquaporin-2 (AQP2) antibody (1:200; a gift from Dr. M. A. Knepper), and goat anti-human Tamm-Horsfall antibody (1:1,000; Organon Technika), which specifically recognize mouse SCD1, AQP2, and Tamm-Horsfall protein. Briefly, the slides were incubated first with 3% H 2 O 2 to eliminate endogenous peroxidase activity and thereafter with antibodies for 60 min. The sections were rinsed with Tris-buffered saline containing 0.1% Tween 20 and a biotinylated secondary antibody against rabbit or goat immunoglobulin for 30 min. Then sections were incubated with horseradish peroxidase (HRP)-conjugated streptavidin for 20 min. HRP labeling was detected by peroxidase substrate solution and counterstained with hematoxylin before being examined under a light microscope.
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Construction of a mouse SCD1 promoter-driven luciferase reporter plasmid. A 716-bp fragment of mouse SCD1 promoter was amplified from mouse embryonic stem cell (ES cell) genomic DNA using a set of primers (upstream primer: 5'-GGTGCCTGGAAGTGGGGGTAG-3' and downstream primer: 5'-CTCTCGGGATGGGTGTTCAGC-3'). The fragment was then cloned into the pGL3-luciferase plasmid (Promega), and the resultant construct designated mSCD1 716-Luc was sequenced to confirm the orientation and sequence. The mouse SCD1 promoter amplified was 100% matched to a published sequence (GenBank accession no. AC123853 ).
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Transient transfections and assays for mSCD1 716-luciferase and SREBP1c (2.6 kb)-luciferase reporter activity. MCT cells, a murine proximal cell line, were kindly provided by Dr. Eric Neilson at Vanderbilt University and were transfected with mSCD1 716-Luc, a reporter construct containing 1 putative SBE [nuclear transcript (nt) -412 to 422; see Ref. 4 ] with or without a constitutive active form of SREBP1c expression vector (SREBP1cN; a gift from Dr. Y. Zhu at the University of California at Riverside), or a rabbit LXR expression vector ( 37 ) using the Effectene Transfection Reagent as recommended by the supplier (Qiagen, Valencia, CA). After incubation for 18 h, the cells were then harvested in 1 x luciferase lysis buffer (Dual Luciferase Kit; Promega), and relative light units were determined using a luminometer (Mono light 2010; Analytical Luminescence Laboratory, San Diego, CA).: w& x/ j1 w5 W' G5 r; F, d2 \- P; o
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To examine the effect of LXR activation on SREBP1c transcription, the MCT cells were transfected with the SREBP1c (2.6 kb)-luciferase reporter (kindly provided by Dr. H. Shimano at the University of Tsukuba; see Ref. 2 ) and then treated with or without TO-901317 (10 µM) for 8 h. A dual luciferase kit was used to detect the light units that represent SREBP1c promoter activity.( r, W8 K* w' y. p$ U% i5 y
5 \! V: p/ S: d& GQuantitative real-time PCR. To assess the SREBP1c and SCD1 mRNA levels in cultured MCT cells, quantitative RT-PCR was used. Briefly, 2 µg of total RNA were used for reverse transcription using Moloney murine leukemia virus RT and 0.4 µM of random primers according to the manufacturer's protocol (Promega reverse transcription system A3500). cDNA samples were then used as templates for quantitative PCR. Primers used were 5'-AAG TTC GCA CTG GCA CCTAC-3' and 5'-AAC CAA GAA GCC CTG GAG AC-3' (mouse tubulin, 182 bp), 5'-CTT CTT GCG ATA CAC TCT GGT-3' and 5'-AGG AAC TCA GAA GCC CAA AGC-3' (mouse SCD1, 352 bp), and 5'-GGA GCC ATG GAT TGC ACA TT-3' and 5'-GCT TCC AGA GAG GAG GCC AG-3' (mouse SREBP1c, 193 bp). Endogenous RNA control tubulin was used for normalization as an internal control.0 |3 D: S) N6 j: P2 n" Q* K
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Western blot analysis. MCT cells were cultured as previously reported ( 14 ). Cells were grown to 80% confluence in six-well plates and treated with or without TO-901317 for 24 h. Cells were scraped off the plates and lysed in ice-cold buffer (20 mM Tris, 140 mM NaCl, 3 mM EDTA, 10 mM NaF, 10 mM sodium pyrophosphate, 2 mM NaVO 4, 10% glycerol, pH 7.4, and 1% Triton X-100) with protease inhibitors (1.5 µM aprotinin, 20 µM leupeptin, 50 µM phenylmethylsulfonyl fluoride, and 1.5 µM benzamidine). The insoluble material was removed by centrifugation at 20,000 g for 30 min at 4°C. Samples containing equal amounts of protein were resolved by SDS-PAGE and transferred to nitrocellulose membranes. After incubation with rabbit anti-SREBP1 antibody at a dilution of 1:200 (catalog no. SC-367; Santa Cruz Biotechnology), the blots were washed and incubated with peroxidase-conjugated secondary antibody, and protein bands were analyzed using a chemiluminescence kit (Santa Cruz Biotechnology).
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LXRs are expressed in mouse kidney. As shown in Fig. 1, LXR mRNA was detected by RT-PCR yielding the expected 328-bp fragments in three individual kidney samples. RT-PCR for LXR mRNA expression also indicated the presence of a single 514-bp band corresponding to the predicted size of the LXR product in all three mouse kidney samples. In addition, both LXR and LXR mRNA were detected in cultured MCT cells, a murine proximal tubule cell line (data not shown).
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Fig. 1. RT-PCR analysis demonstrating both liver X receptor (LXR)- and LXR mRNA was expressed in normal mouse kidneys. Total RNA was isolated from the kidneys collected from 3 male C57Bl/6 mice and treated with RNase-free DNase I. RT-PCR was performed as described in MATERIALS AND METHODS. The arrows indicate bands of LXR (328 bp; A ), LXR (514 bp; B ), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 178 bp; C ) cDNA fragments amplified by PCR. GAPDH was used as an RNA loading control.. T$ n" h5 ?# k$ W9 A0 W
% r) \3 R1 h3 v% u: bEffect of LXR agonist TO-901317 on gene expression profile in the kidney. The renal gene expression profile regulated by the LXR agonist TO-901317 was examined using the Affymetrix GeneChip Microarray. Of the 51 genes differentially expressed in the kidneys from the mice treated with or without TO-901317, 11 known genes were upregulated by TO-901317 and 40 genes were downregulated. One of the most striking changes revealed by GeneChip was the induction of a group of genes that are involved in catalyzing the synthesis of fatty acids, including SCD1, SCD2, and SREBP1 ( Table 1 ). SCD1 and SCD2 mRNA were expressed at much higher levels in the kidneys of mice treated with LXR agonist TO-901317 compared with vehicle-treated animals. Based on Affymetrix analysis, SCD1 and SCD2 were upregulated by 8-fold and 2.5-fold in TO-901317-treated mice, respectively ( Table 1 ).
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. H# b) x* p, k8 \9 ^Table 1. A partial list of genes regulated by LXR agonist TO-901317 treatment in mouse kidney
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2 v9 S7 c# e6 m# _Induction of SCD1 and SCD2 gene expression in mouse kidney by TO-901317. The LXR agonist TO-901317 markedly increased renal expression of SCD1 and SCD2 gene in the mouse. As shown in Fig. 2 A, Northern blot analysis confirmed a marked increase in both SCD1 and SCD2 gene expression. Consistent with the data from the Affymetrix study, quantitative analysis further revealed 5.4- and 2.9-fold upregulation of SCD1 and SCD2 genes in mouse kidneys by TO-901317 treatment, respectively ( Fig. 2 B ).' M+ l' f: W7 Q
7 a. e" t. _6 f0 tFig. 2. Northern blot analysis showing the LXR agonist TO-901317 significantly increased stearoyl-CoA desaturase (SCD)-1 and SCD2 mRNA expression in mouse kidney. Animals were treated as described under MATERIALS AND METHODS. A : renal SCD1 and SCD2 mRNA levels in control and TO-901317-treated mice. Total RNA was fragmented by agarose electrophoresis and blotted to nylon membranes. The blots were then hybridized with a specific cDNA probe for mouse SCD1 or SCD2 and washed under high stringency. Loading and integrity of RNA is shown with ethidium bromide staining ( bottom ). B : quantitative analysis of SCD1 mRNA expression in the kidneys. The degree of change in the TO-901317-treated group relative to that of the untreated group was calculated based on the signals of densitometric scanning of the Northern blot. * P
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Increased fatty acid desaturation index in the kidneys by TO-901317 treatment. To assess the effect of TO-901317 on SCD activity in the kidney, the long-chain fatty acid profile was analyzed. As shown in Fig. 3, SCD1 desaturation indexes as reflected by oleate palmitoleate/stearate palmitate (C16:01 C18:01/C16:0 C18:0) in the triglyceride fraction of the kidneys of mice treated by TO-901317 were significantly greater than the control mice ( P
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Fig. 3. LXR agonist TO-901317 increased the ratio of monounsaturated to saturated fatty acids in the renal triglyceride fraction (the desaturation index). The fatty acid profile was measured by gas-liquid chromatography. Data were expressed as the ratio of monounsaturated to saturated long-chain fatty acids for oleate palmitoleate/stearate palmitate (C16:01 C18:01/C16:0 C18:0) in the triglyceride fraction. * P
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Intrarenal localization of SCD1. In situ hybridization was used to define the intrarenal expression of SCD1 mRNA. In both vehicle- and TO-901317-treated mice, SCD1 mRNA was predominantly expressed in the outer stripe of the outer medulla. TO-901317 treatment for 3 days dramatically increased SCD1 mRNA expression in this region ( Fig. 4 ).3 ~2 ^) V3 f0 N6 [' K* Q1 [' ?9 L
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Fig. 4. Autoradiograph of in situ hybridization analysis demonstrating SCD1 mRNA expression in mouse kidneys. Radiolabed mouse SCD1 antisense riboprobe (352 bp) was used. Black grains indicate hybridization signals of SCD1 mRNA. Much more intensive SCD1 mRNA signals were observed in TO-901317-treated mouse kidneys ( right ) than that in vehicle-treated mouse kidneys ( left ). Note: SCD1 mRNA was also highly expressed in perirenal adipose tissues. No specific signals were detected using sense SCD1 riboprobe (data not shown).0 ?$ b/ M; i* G1 m- ?
% f+ K/ v! P; [$ b$ w, hIn agreement with the result from in situ hybridization analysis, immunohistochemical studies confirmed this pattern of intrarenal localization of SCD1. SCD1 immunoreactivity was observed in a subset of tubular structures in the region of the outer medulla in mouse kidney ( Fig. 5 ). The localization, size, and cuboidal shape of epithelial cells expressing SCD1 protein suggest SCD1 may be expressed in the proximal straight tubule cells, medullary collecting ducts, or medullary thick ascending limb cells rather than the cells in the thin limb of the loop of Henle and the vasa recta ( Fig. 5 ).
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Fig. 5. Immunohistochemical studies showing intrarenal SCD1 protein expression in vehicle ( middle )- and TO-901317-treated ( right ) mice. Mice were administrated with vehicle or LXR agonist TO-901317 (3 mg·kg -1 ·day -1 ) for 3 days. Note: immunoreactivity of SCD1 protein (brown color) was observed in the outer stripe of the outer medulla in both vehicle- and TO-901317-treated mice ( B and C, magnification x 50). The higher magnification demonstrates SCD1 immunoreactivity was only localized in a subset of tubular structures ( E and F, magnification x 400). In the kidneys of mice receiving TO-901317 treatment ( C and F ), SCD1 immunoreactivity was much higher than that in vehicle-treated control animals ( B and E ). Note: no positive SCD1 immunostaining was observed in absence of SCD1 antibody in control normal mouse kidney ( A and D ).
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9 k+ s7 N0 i2 n- F" W. xSegmental expression of SCD1 mRNA. To further define the tubule segments expressing SCD1, double-labeling studies using both in situ hybridization and immunostaining with segmental specific markers were performed. Anti-AQP2 antibody was used to specifically stain medullary collecting ducts ( Fig. 6 ). SCD1 mRNA expression as assessed by in situ hybridization was not expressed in AQP2-positive renal tubules. Double-labeling studies using in situ hybridization for SCD1 mRNA and immunohistochemistry for Tamm-Horsfall protein also showed that SCD1 gene expression was not colocalized with Tamm-Horsfall-positive tubules ( Fig. 7 ). These findings suggest that SCD1 was expressed in proximal straight tubule.
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Fig. 6. Double-labeling studies demonstrating SCD1 mRNA was expressed in aquaporin-2 (AQP2)-negative renal tubules in mouse kidneys. Kidney sections from TO-901317-treated animals were subjected to in situ hybridization of SCD1 mRNA and immunohistochemical examination of AQP2 protein. Black dots and brown color represent SCD1 mRNA hybridization signals and AQP2 immunoreactivity, respectively. Note that SCD1 mRNA expression (pink arrows) did not overlap with AQP2 immunoreactivity in collecting duct (red arrows). Glom, glomerulus. Magnification x 400.
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% Y2 } M7 G7 V2 b Q: m# D8 x% O0 M0 GFig. 7. In situ hybridization and immunohistochemistry studies showing SCD1 mRNA expression in Tamm-Horsfall (T-H)-negative renal tubules. In situ hybridization analysis of SCD1 mRNA and immunohistochemical study of Tamm-Horsfall protein were performed in the kidneys of TO-901317-treated mice as described under MATERIALS AND METHODS. Black grains and brown color represent SCD1 mRNA signals and Tamm-Horsfall immunoreactivity, respectively. Note SCD1 mRNA expression did not overlap with Tamm-Horsfall immunostaining in medullary thick ascending limb (mTAL). Magnification x 400.8 ]5 O( _' H; a! }/ F
U4 \3 L. U2 e3 g+ B9 L% g/ n8 JLXR activation induced SREBP1c expression in MCT cells. Sequence analysis failed to reveal whether a putative LXRE consensus site exists in the mouse SCD1 promoter region (TESS software http://agave.upenn.edu/tess/index.html ), suggesting SCD1 may not be a direct transcriptional target of LXRs. To determine the mechanisms by which LXR agonist TO-901317 upregulated SCD1 expression in renal proximal tubule cells, MCT cells were cultured and treated with TO-901317 (10 µM for 24 h) to examine the effect of LXR activation on SREBP1c expression. Consistent with the findings revealed by the Affymetrix gene chip analysis ( Table 1 ), LXR activation markedly increased the levels of the 68-kDa NH 2 -terminal fragment that is responsible for activating SREBP1c target gene transcription ( Fig. 8 A ). In support of this, activation of LXRs by TO-901317 treatment significantly enhanced mouse SREBP1c promoter activity ( Fig. 8 B ) and resulted in a 3.5-fold increase in SREBP1c mRNA expression, as assessed by quantitative real-time PCR ( P 9 r; L) U6 X/ }) H% J
/ B" W. i. w' ^( z* V. ]1 O* \Fig. 8. Effect of LXR activation on sterol regulatory element-binding protein (SREBP)-1c expression in cultured murine proximal tubule (MCT) cells. A : induction of SREBP1c expression by TO-901317 treatment in MCT cells. Cells were treated with TO-901317 (10 µM) for 24 h. Active cleavage products of SREBP1c (68 kDa) were examined using immunoblot assay. -Actin was served as a protein-loading control. B : LXR activator TO-901317 significantly increased mouse SREBP1c promoter activity. Mouse SREBP1c reporter plasmid, SREBP1c (2.6 kb)-luciferase, was transfected in MCT cells and then treated with or without TO-901317 (10 µM) for 8 h. * P ! x, [( E1 l# o9 w% k4 t- [
6 r# R- E5 c0 ]- T( C; e' R+ l7 lSREBP1c increased mouse SCD1 promoter activity. Consistent with a previous report ( 4 ), sequence analysis of our cloned 716 bp of mouse SCD1 promoter region by the TESS software revealed a putative SREBP binding site (SRE; nt -412 to 422; Fig. 9 A ). Cotransfection of MCT cells with a constitutively active SREBP1 expression vector (SREBP1cN) significantly increased mouse SCD1 promoter activity ( Fig. 9 B ). However, cotransfection of a LXR expression vector had little effect on SCD1 transcription (data not shown), further supporting the idea that enhanced SREBP1c expression may be responsible for TO-901317-mediated SCD1 induction. d% O v7 X. |, Y1 x
5 Y% J+ a2 D# e3 ^+ u2 ^Fig. 9. SREBP1c increased mouse SCD1 promoter activity. A: existence of SREBP-binding element (SRE) in mouse SCD1 promoter (nuclear transcript -412 to -422). B : overexpression of an active SREBP1c expression vector significantly increased mouse SCD1 promoter activity. mSCD1 716-Luc plasmid was transfected with or without a SREBP1cN in MCT cells, and luciferase activity was measured. Note: cotransfection of SREBP1cN significantly increased SCD1 transcription. NF-Y, nuclear factor-Y. ** P 0 d- i9 h3 f+ u0 n
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LXRs are nuclear receptor transcription factors and play an important role in cholesterol metabolism and lipid biosynthesis ( 15 ). Although LXRs are predominantly expressed in adipose tissue and liver, moderate levels of LXR and LXR were also found in the kidney ( 17 ). To date, little is known about the biological function of LXR receptors in the kidney. The present studies reveal for the first time that an LXR agonist significantly increases expression of SCD1 in renal proximal straight tubules of mouse ( Fig. 10 ). SCD1 is the rate-limiting enzyme catalyzing the conversion of saturated long-chain fatty acids into monounsaturated fatty acids (MUFAs; see Refs. 22 and 23 ); thus, LXRs may be involved in regulating lipid metabolism, especially in biosynthesis of triglycerides, cholesterol esters, and phospholipids in this nephron segment.
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Fig. 10. A : schematic illustration of intrarenal localization of SCD1 in the proximal straight tubules (PST, black arrow) of mouse kidney. SCD1 was expressed in the outer stripe (OS) of the outer medulla and was not colocalized with AQP2-positive (magenta arrow) and Tamm-Horsfall-positive (yellow arrow) renal tubules. The cubic shape of cells expressing SCD1 excluded the endothelial cells of vasa recta (red arrow) and epithelial cells of thin limbs of Henle's loop. IS, inner stripe. B: mechanisms involved in LXR activation-induced SCD1 gene expression in renal proximal tubules. MCD, medullary collecting duct.
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Our results demonstrated that both LXR and LXR are expressed in mouse kidney, suggesting these receptors may play an important role in regulating renal physiology. We and others ( 30, 37 ) have previously reported that LXR was highly expressed in rabbit kidney, with wide expression in every segment along the nephron including the glomeruli, proximal tubules, and cultured glomerular mesangial cells. Activation of LXR in mesangial cells resulted in marked induction of gene transcription of ABCA1 ( 30 ), a membrane-associated transporter mediating cholesterol efflux ( 18 ). These findings suggest LXR may be an important modulator in regulating lipid metabolism in the kidney, especially in maintaining intraglomerular lipid homeostasis ( 37 ). Consistent with previous reports in which LXR was found to be widely expressed in late stages of embryonic kidney development ( 3, 17 ), the present study also indicated the LXR receptor is expressed in adult kidney. Although, in general, no clear role for this receptor has yet been identified, expression of LXR at high levels in developing and adult organs, including brain, thyroid gland, adrenal, testis, ovary, and kidney, suggests LXR may be involved in the morphogenesis or physiology of these tissues ( 3, 17, 37 ).) T6 ?3 L0 d* T$ p0 y2 x2 h& w+ y
+ F' U4 K/ W' }3 y1 l' q& ^$ DTo gain further insight into the role of LXR and LXR in renal function, gene expression profiling using microarrays (mouse-specific array; Affymetrix) was employed for the investigation of the effect of LXR agonist TO-901317 on renal gene expression. Among 51 genes differentially regulated by LXRs in the kidney, a group of genes involved in catalyzing the synthesis of fatty acids and converting the saturated fatty acids into MUFAs were found to be most markedly increased. These genes include SREBP1, SCD1, and SCD2. SREBP1 is a membrane-bound transcription factor and has been postulated to be a master regulator in the synthesis of fatty acids, cholesterol, and triglycerides ( 1, 11 ) and a direct target gene of LXR and LXR ( 29 ). The mammalian genome contains only one SREBP1 gene, but as a result of alternative transcription start sites, two SREBP1 isoforms exist, designated SREBP1a and SREBP1c ( 5 ). SREBP1a is a potent activator of all SREBP response genes and enhances both fatty acid and cholesterol synthesis, whereas the role of SREBP1c is more restricted and it preferentially participates in fatty acid biosynthesis ( 11 ). Although SREBP1 is highly expressed in the liver, steroidogenic organs, and adipose tissue, significant levels of SREBP1a and -1c are also found to be present in the kidney ( 33 ). The induction of SREBP1c gene expression after LXR activation in the kidney raises the possibility that, in addition to liver and adipose tissue, LXRs may also regulate renal lipid metabolism through activating a SREBP1 transcriptional pathway. At present, it remains unclear which nephron segments are significantly affected by LXR ( 30, 31 ). Such information will be fundamental to understand the LXR-modulated lipid homeostasis in the kidney.
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The present studies also demonstrated a marked induction of a group of genes that mediate the conversion of saturated fatty acids into MUFAs, including SCD1 and SCD2 in the kidney after LXR agonist treatment. SCDs are the rate-limiting enzymes catalyzing the synthesis of oleate (18:1) and palmitoleate (C16:1) from stearate (C18:0) and palmitate (C16:0), respectively ( 24 ). Oleate and palmitoleate, the main product of SCDs, represent the major MUFAs of membrane phospholipids, triglycerides, and cholesterol esters, and the ratio of saturated fatty acid to MUFAs affects phospholipids composition and contributes to the pathogenesis of many diseases, including atherosclerosis, obesity, and diabetes ( 23 ). Studies in genetically engineered mice clearly indicate that SCD1 activity is reversely associated with insulin sensitivity and adiposity and correlates with triglyceride and cholesterol ester biosynthesis in vivo ( 7, 8, 25, 27 ). Thus the induction of SCD1 and SCD2 gene expression and enzymatic activity by LXR activation may alter the composition of membrane phospholipids and triglyceride and cholesterol ester content in the kidney, thereby affecting renal function.* @+ h& g8 o0 `7 ]3 H9 p0 l' a
% O/ l* ^1 }3 @& y0 ?Reduced SCD1 expression, which is associated with decreased fatty acid synthesis and increased fatty acid oxidation in primary hepatocytes ( 12 ), was previously reported in the kidney of diabetic NOD mice ( 36 ), suggesting SCD1 may play an important role in pathogenesis of diabetic nephropathy. The present studies provide the first in vivo evidence that renal SCD1 gene expression is differentially increased in the proximal straight tubules by an LXR agonist. Because SCD1 catalyzes the 9- cis desaturation of saturated fatty acyl-CoAs, including palmitoyl- and stearoyl-CoA, its activation by LXR agonist may increase the ratio of oleate to palmitoleate to stearate to palmitate in epithelial cells of the proximal straight tubules, thereby improving cell membrane fluidity and enhancing cell function and integrity ( 22 ). In addition, increased expression of SCD1 in proximal straight tubules by LXR agonists may indirectly protect these cells from harmful effects of free cholesterol and free saturated fatty acids by converting them to cholesterol ester and triglyceride ( 13, 16, 21 ). It has been previously shown that excessive intracellular free cholesterol induces a number of cellular events, including cholesterol crystallization and cell death ( 1, 13 ), and increased SCD1 activity promotes desaturation of saturated fatty acids and enhances the synthesis of less toxic cholesterol ester ( 21 ). With respect to fatty acids, excess unsaturated oleic acid leads to triglyceride accumulation and is well tolerated, whereas overload-saturated palmitic acid caused marked apoptosis and is poorly incorporated into triglyceride ( 16 ). In addition, enhanced SCD1 expression has been reported to be associated with an increase in cholesterol efflux to HDL2 ( 35 ), possibly due to more unsaturated lipid composition ( 19 ). Considered together, the present studies suggest the LXRs could play an important role in maintaining the lipid homeostasis in renal proximal straight tubules by promoting fatty acid desaturation. Increased SCD1 expression may lead to enhanced intracellular levels of unsaturated fatty acids ( 35 ), thereby serving a protective function against lipotoxicity through promotion of triglyceride and cholesterol ester biosynthesis and passive efflux of free cholesterol.6 X4 W5 N; D' H! S& R6 a
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At present, little is known about the mechanisms by which TO-901317 upregulates SCD1 gene expression in the proximal straight tubules. Sequence analysis of 716 bp of mouse SCD1 promoter sequence revealed no LXRE exists in this region, suggesting SCD1 gene transcription is not under direct control of LXR receptors. In support of this, overexpression of an LXR expression vector did not induce SCD1-luciferase activity. Among multiple genes induced by TO-901317 in the kidney, SREBP1 is a direct target gene of LXRs ( 38 ) and a key regulator of many genes involved in lipid metabolism, including SCD1 ( 4, 28 ). SCD1 is reported to be directly regulated by SREBP1c via binding to the SREBP binding element (SRE) in its promoter region ( 4 ). In the present studies, we demonstrated that TO-901317 enhanced SREBP1c transcription and increased active SREBP1c (SREBP1cN) expression in cultured renal proximal tubule cells, which may contribute to increased SCD1 expression in these cells.9 H5 a0 ^5 n* f, P9 j1 m
$ U! N6 X" {; K7 sIn conclusion, pharmacological activation of LXRs by TO-901317 results in marked upregulation of SCD1 gene expression via the SREBP1c pathway in renal proximal straight tubule cells ( Fig. 9 ), where enhanced SCD1 activity may mediate fatty acid desaturation and therefore modulate cellular function and lipid homeostasis." d P4 M5 F3 {5 u! K/ D
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0 \/ _8 [0 p0 n7 N0 c& H7 tThe study was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grants RO1 DK-065074-02 and P01-DK-38226 (to M. D. Breyer), the Natural Science Foundation and the Ministry of Education of China (30271521/30530340/104001 and Program for Changjiang Scholars and Innovative Research Team), and the Genzyme Renal Innovation Program (to Y. Guan).. i! E, |/ O4 K+ f
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发表于 2009-4-22 08:45
