Renal Ischemia-Induced Cholesterol Loading
2008; Elsevier BV; Volume: 174; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2009.080602
ISSN1525-2191
AutoresMasayo Naito, Karol Bomsztyk, Richard A. Zager,
Tópico(s)Drug Transport and Resistance Mechanisms
ResumoAcute kidney injury evokes renal tubular cholesterol synthesis. However, the factors during acute kidney injury that regulate HMG CoA reductase (HMGCR) activity, the rate-limiting step in cholesterol synthesis, have not been defined. To investigate these factors, mice were subjected to 30 minutes of either unilateral renal ischemia or sham surgery. After 3 days, bilateral nephrectomy was performed and cortical tissue extracts were prepared. The recruitment of RNA polymerase II (Pol II), transcription factors (SREBP-1, SREBP-2, NF-κB, c-Fos, and c-Jun), and heat shock proteins (HSP-70 and heme oxygenase-1) to the HMGCR promoter and transcription region (start/end exons) were assessed by Matrix ChIP assay. HMGCR mRNA, protein, and cholesterol levels were determined. Finally, histone modifications at HMGCR were assessed. Ischemia/reperfusion (I/R) induced marked cholesterol loading, which corresponded with elevated Pol II recruitment to HMGCR and increased expression levels of both HMGCR protein and mRNA. I/R also induced the binding of multiple transcription factors (SREBP-1, SREBP-2, c-Fos, c-Jun, NF-κB) and heat shock proteins to the HMGCR promoter and transcription regions. Significant histone modifications (increased H3K4m3, H3K19Ac, and H2A.Z variant) at these loci were also observed but were not identified at either the 5′ and 3′ HMGCR flanking regions (±5000 bps) or at negative control genes (β-actin and β-globin). In conclusion, I/R activates the HMGCR gene via multiple stress-activated transcriptional and epigenetic pathways, contributing to renal cholesterol loading. Acute kidney injury evokes renal tubular cholesterol synthesis. However, the factors during acute kidney injury that regulate HMG CoA reductase (HMGCR) activity, the rate-limiting step in cholesterol synthesis, have not been defined. To investigate these factors, mice were subjected to 30 minutes of either unilateral renal ischemia or sham surgery. After 3 days, bilateral nephrectomy was performed and cortical tissue extracts were prepared. The recruitment of RNA polymerase II (Pol II), transcription factors (SREBP-1, SREBP-2, NF-κB, c-Fos, and c-Jun), and heat shock proteins (HSP-70 and heme oxygenase-1) to the HMGCR promoter and transcription region (start/end exons) were assessed by Matrix ChIP assay. HMGCR mRNA, protein, and cholesterol levels were determined. Finally, histone modifications at HMGCR were assessed. Ischemia/reperfusion (I/R) induced marked cholesterol loading, which corresponded with elevated Pol II recruitment to HMGCR and increased expression levels of both HMGCR protein and mRNA. I/R also induced the binding of multiple transcription factors (SREBP-1, SREBP-2, c-Fos, c-Jun, NF-κB) and heat shock proteins to the HMGCR promoter and transcription regions. Significant histone modifications (increased H3K4m3, H3K19Ac, and H2A.Z variant) at these loci were also observed but were not identified at either the 5′ and 3′ HMGCR flanking regions (±5000 bps) or at negative control genes (β-actin and β-globin). In conclusion, I/R activates the HMGCR gene via multiple stress-activated transcriptional and epigenetic pathways, contributing to renal cholesterol loading. Diverse forms of tissue injury evoke cellular responses that confer protection against subsequent ischemic or toxic attack. This adaptation has been denoted by heterogeneous terms that include: acquired cytoresistance, the stunning phenomenon, the heat shock response, and ischemic preconditioning. It has been recognized for a century that the kidney can undergo this same injury adaptation.1Honda N Hishida A Ikuma K Yonemura K Acquired resistance to acute renal failure.Kidney Int. 1987; 31: 1233-1238Crossref PubMed Scopus (84) Google Scholar This is based on observations that exposing the kidney to one nephrotoxin elicits protection against the same, or a different (cross resistance), nephrotoxic agent.1Honda N Hishida A Ikuma K Yonemura K Acquired resistance to acute renal failure.Kidney Int. 1987; 31: 1233-1238Crossref PubMed Scopus (84) Google Scholar In 1984, this laboratory demonstrated that this phenomenon is not restricted to nephrotoxic injury: when mild ischemic renal injury was induced in rats, dramatic protection against subsequent, and more severe, renal ischemia resulted.2Zager RA Baltes LA Sharma HM Jurkowitz MS Responses of the ischemic acute renal failure kidney to additional ischemic events.Kidney Int. 1984; 26: 689-700Crossref PubMed Scopus (105) Google Scholar Indeed, this was probably the first demonstration of the so-called ischemic preconditioning phenomenon, subsequently confirmed in many extra-renal tissues (eg, heart, liver, intestine, brain).3Reimer KA Murry CE Jennings RB Cardiac adaptation to ischemia. 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As noted above, this same preconditioning develops after toxic renal damage. Furthermore, nonischemic/nontoxic insults can also induce the renal cytoresistant state. 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A novel form of acquired, heme oxygenase-dependent resistance to renal injury.J Clin Invest. 1996; 98: 2139-2145Crossref PubMed Scopus (115) Google Scholar Therefore, the phenomenon of ischemic preconditioning is best considered to be one of any number of stressors that can elicit subsequent protection against acute renal failure. There are undoubtedly multiple pathways by which injured renal tubular cells acquire resistance to further damage. The one that has been most widely recognized is the induction of cytoprotective stress proteins, such as the heat shock protein-70 (HSP-70), HSP-32 (ie, heme oxygenase-1; HO-1), and ferritin.8Jo SK Ko GJ Boo CS Cho WY Kim HK Heat preconditioning attenuates renal injury in ischemic ARF in rats: role of heat-shock protein 70 on NF-kappa B-mediated inflammation and on tubular cell injury.J Am Soc Nephrol. 2006; 17: 3082-3092Crossref PubMed Scopus (54) Google Scholar, 9Riordan M Sreedharan R Kashgarian M Siegel NJ Modulation of renal cell injury by heat shock proteins: lessons learned from the immature kidney.Nat Clin Pract Nephrol. 2006; 2: 149-156Crossref PubMed Scopus (9) Google Scholar, 20Balla J Nath KA Balla G Juckett MB Jacob HS Vercellotti GM Endothelial cell heme oxygenase and ferritin induction in rat lung by hemoglobin in vivo.Am J Physiol. 1995; 268: L321-L327PubMed Google Scholar, 21Berenshtein E Vaisman B Goldberg-Langerman C Kitrossky N Konijn AM Chevion M Roles of ferritin and iron in ischemic preconditioning of the heart.Mol Cell Biochem. 2002; 234: 283-292Crossref PubMed Scopus (50) Google Scholar Alterations in lipid homeostasis may also be involved. For example, arachidonic acid release from phospholipids,22Zager RA Schimpf BA Gmur DJ Burke TJ Phospholipase A2 can protect renal tubules from oxygen deprivation injury.Proc Natl Acad Sci USA. 1993; 90: 8297-8301Crossref PubMed Scopus (39) Google Scholar, 23Alkhunaizi AM Yaqoob MM Edelstein CL Gengaro PE Burke TJ Nemenoff RA Schrier RW Arachidonic acid protects against hypoxic injury in rat proximal tubules.Kidney Int. 1996; 49: 620-625Crossref PubMed Scopus (26) Google Scholar, 24Zager RA Conrad DS Burkhart K Phospholipase A2: a potentially important determinant of adenosine triphosphate levels during hypoxic-reoxygenation tubular injury.J Am Soc Nephrol. 1996; 7: 2327-2339PubMed Google Scholar as well as sphingomyelin-generated sphingolipid products,25Jin ZQ Karliner JS Vessey DA Ischaemic postconditioning protects isolated mouse hearts against ischaemia/reperfusion injury via sphingosine kinase isoform-1 activation.Cardiovasc Res. 2008; 79: 134-140Crossref PubMed Scopus (74) Google Scholar, 26Iwata M Herrington J Zager RA Sphingosine: a mediator of acute renal tubular injury and subsequent cytoresistance.Proc Natl Acad Sci USA. 1995; 92: 8970-8974Crossref PubMed Scopus (54) Google Scholar, 27Nishino Y Webb I Marber MS Sphingosine kinase isoforms and cardiac protection.Cardiovasc Res. 2007; 76: 3-4Crossref PubMed Scopus (6) Google Scholar can evoke a cytoprotective response. Probably the most consistent, and stable, renal injury-induced lipid alteration is renal tubular cholesterol accumulation.15Zager RA Johnson AC Lund S 'Endotoxin tolerance': TNF-alpha hyper-reactivity and tubular cytoresistance in a renal cholesterol loading state.Kidney Int. 2007; 71: 496-503Crossref PubMed Scopus (32) Google Scholar, 28Zager RA Burkhart KM Johnson ACM Sacks BM Increased proximal tubular cholesterol content: implications for cell injury and "acquired cytoresistance.".Kidney Int. 1999; 56: 1788-1797Crossref PubMed Scopus (69) Google Scholar, 29Zager RA Kalhorn TF Changes in free and esterified cholesterol; hallmarks of acute renal tubular injury and acquired cytoresistance.Am J Pathol. 2000; 157: 1007-1016Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 30Zager RA Plasma membrane cholesterol: a critical determinant of cellular energetics and tubular resistance to attack.Kidney Int. 2000; 58: 193-205Crossref PubMed Scopus (56) Google Scholar, 31Zager RA Johnson A Renal cortical cholesterol accumulation is an integral component of the systemic stress response.Kidney Int. 2001; 60: 2299-2310Crossref PubMed Scopus (49) Google Scholar, 32Zager RA P glycoprotein-mediated cholesterol cycling determines proximal tubular cell viability.Kidney Int. 2001; 60: 944-956Crossref PubMed Scopus (12) Google Scholar, 33Zager RA Andoh T Bennett WM Renal cholesterol accumulation: a durable response after acute and subacute renal insults.Am J Pathol. 2001; 159: 743-752Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 34Zager RA Shah VO Shah HV Zager PG Johnson ACM Hanson S The mevalonate pathway during acute tubular injury.Am J Pathol. 2002; 161: 681-692Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar For example, in each of the above-mentioned models of renal cytoresistance (IR; myoglobinuric acute renal failure, glomerulonephritis, nephrotoxins, heat shock, ureteral obstruction, endotoxemia), an ∼25 to 50% sustained increase in renal tubular cholesterol concentrations result.28Zager RA Burkhart KM Johnson ACM Sacks BM Increased proximal tubular cholesterol content: implications for cell injury and "acquired cytoresistance.".Kidney Int. 1999; 56: 1788-1797Crossref PubMed Scopus (69) Google Scholar, 29Zager RA Kalhorn TF Changes in free and esterified cholesterol; hallmarks of acute renal tubular injury and acquired cytoresistance.Am J Pathol. 2000; 157: 1007-1016Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 30Zager RA Plasma membrane cholesterol: a critical determinant of cellular energetics and tubular resistance to attack.Kidney Int. 2000; 58: 193-205Crossref PubMed Scopus (56) Google Scholar, 31Zager RA Johnson A Renal cortical cholesterol accumulation is an integral component of the systemic stress response.Kidney Int. 2001; 60: 2299-2310Crossref PubMed Scopus (49) Google Scholar, 32Zager RA P glycoprotein-mediated cholesterol cycling determines proximal tubular cell viability.Kidney Int. 2001; 60: 944-956Crossref PubMed Scopus (12) Google Scholar, 33Zager RA Andoh T Bennett WM Renal cholesterol accumulation: a durable response after acute and subacute renal insults.Am J Pathol. 2001; 159: 743-752Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 34Zager RA Shah VO Shah HV Zager PG Johnson ACM Hanson S The mevalonate pathway during acute tubular injury.Am J Pathol. 2002; 161: 681-692Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar The precise mechanism(s) by which excess cholesterol exerts its protective action remains to be defined. However, the available data suggest that cholesterol increases plasma membrane and mitochondrial membrane rigidity, and this serves to maintain mitochondrial energetics and plasma membrane integrity during superimposed ischemic or toxic attack.28Zager RA Burkhart KM Johnson ACM Sacks BM Increased proximal tubular cholesterol content: implications for cell injury and "acquired cytoresistance.".Kidney Int. 1999; 56: 1788-1797Crossref PubMed Scopus (69) Google Scholar, 30Zager RA Plasma membrane cholesterol: a critical determinant of cellular energetics and tubular resistance to attack.Kidney Int. 2000; 58: 193-205Crossref PubMed Scopus (56) Google Scholar, 32Zager RA P glycoprotein-mediated cholesterol cycling determines proximal tubular cell viability.Kidney Int. 2001; 60: 944-956Crossref PubMed Scopus (12) Google Scholar Of note, after injury cholesterol accumulation and cytoresistance are not renal-specific phenomena. For example, we have observed that when acute myelogenous leukemia cells are exposed to cancer chemotherapeutic agents, cholesterol accumulates, conferring resistance to further chemotherapy.35Banker DE Mayer SJ Li HY Willman CL Appelbaum FR Zager RA Cholesterol synthesis and import contribute to protective cholesterol increments in acute myeloid leukemia cells.Blood. 2004; 104: 1816-1824Crossref PubMed Scopus (79) Google Scholar, 36Stirewalt DL Appelbaum FR Willman CL Zager RA Banker DE Mevastatin can increase toxicity in primary AMLs exposed to standard therapeutic agents, but statin efficacy is not simply associated with ras hotspot mutations or over expression.Leuk Res. 2003; 27: 133-145Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 37Li HY Appelbaum FR Willman CL Zager RA Banker DE Cholesterol-modulating agents kill acute myeloid leukemia cells and sensitize them to therapeutics by blocking adaptive cholesterol responses.Blood. 2003; 101: 3628-3634Crossref PubMed Scopus (161) Google Scholar Therefore, the mechanisms that are responsible for injury-induced cholesterol accumulation likely have broad-based biological relevance. Evidence gathered to date indicates that increased tubular cell cholesterol synthesis can contribute to the cholesterol loading state after injury. This assertion is based on observations that statin-mediated inhibition of HMG CoA reductase, the rate-limiting enzyme in cholesterol synthesis, abrogates cholesterol accumulation after injury.31Zager RA Johnson A Renal cortical cholesterol accumulation is an integral component of the systemic stress response.Kidney Int. 2001; 60: 2299-2310Crossref PubMed Scopus (49) Google Scholar, 35Banker DE Mayer SJ Li HY Willman CL Appelbaum FR Zager RA Cholesterol synthesis and import contribute to protective cholesterol increments in acute myeloid leukemia cells.Blood. 2004; 104: 1816-1824Crossref PubMed Scopus (79) Google Scholar, 36Stirewalt DL Appelbaum FR Willman CL Zager RA Banker DE Mevastatin can increase toxicity in primary AMLs exposed to standard therapeutic agents, but statin efficacy is not simply associated with ras hotspot mutations or over expression.Leuk Res. 2003; 27: 133-145Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 37Li HY Appelbaum FR Willman CL Zager RA Banker DE Cholesterol-modulating agents kill acute myeloid leukemia cells and sensitize them to therapeutics by blocking adaptive cholesterol responses.Blood. 2003; 101: 3628-3634Crossref PubMed Scopus (161) Google Scholar, 38Zager RA Johnson AC Naito M Bomsztyk K Maleate nephrotoxicity: mechanisms of injury and correlates with ischemic/hypoxic tubular cell death.Am J Physiol. 2008; 294: F187-F197Crossref PubMed Scopus (50) Google Scholar, 39Zager RA Johnson AC Hanson SY Proximal tubular cholesterol loading after mitochondrial, but not glycolytic, blockade.Am J Physiol. 2003; 285: F1092-F1099PubMed Google Scholar However, whether increased cholesterol synthesis reflects increased HMGCR gene transcription, and which transcription factors might stimulate this response, have not been assessed. Hence, the present study was undertaken to explore the following issues: i) Does renal ischemic preconditioning activate the HMGCR gene (as assessed by RNA polymerase II recruitment to its transcription sites)? ii) If so, which transcription factor(s) might be involved? iii) Do epigenetic modifications exist at the HMGCR gene, potentially facilitating an increased transcriptional state? Investigations into each of these issues form the basis of this study. Male CD 1 mice (30 to 35 g; Charles River Laboratories, Wilmington, MA), maintained under routine vivarium conditions and subjected to Institutional Animal Care and Use Committee approved protocols were used for all experiments. They were subjected to 30 minutes of left renal pedicle occlusion performed through an abdominal incision under pentobarbital anesthesia (40 to 50 mg/kg; IP) and followed by two-layer abdominal wall suturing. Approximately 72 hours after surgery, they were re-anesthetized, and bilateral nephrectomy was performed. The renal cortices were dissected (4°C) and subjected to either lipid, protein, or RNA extraction or chromatin cross linking (formalin).40Naito M Bomsztyk K Zager RA Endotoxin mediates recruitment of RNA polymerase II to target genes in acute renal failure.J Am Soc Nephrol. 2008; 19: 1321-1330Crossref PubMed Scopus (53) Google Scholar Lipid extracts were analyzed for free and esterified cholesterol levels by gas chromatography (expressed as nmol/μmol phospholipid phosphate).29Zager RA Kalhorn TF Changes in free and esterified cholesterol; hallmarks of acute renal tubular injury and acquired cytoresistance.Am J Pathol. 2000; 157: 1007-1016Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 40Naito M Bomsztyk K Zager RA Endotoxin mediates recruitment of RNA polymerase II to target genes in acute renal failure.J Am Soc Nephrol. 2008; 19: 1321-1330Crossref PubMed Scopus (53) Google Scholar Protein samples were probed for HMGCR protein by Western blotting/chemiluminescence.31Zager RA Johnson A Renal cortical cholesterol accumulation is an integral component of the systemic stress response.Kidney Int. 2001; 60: 2299-2310Crossref PubMed Scopus (49) Google Scholar HMGCR mRNA was quantified by competitive polymerase chain reaction (PCR) (expressed as a ratio to simultaneously measured GAPDH product).34Zager RA Shah VO Shah HV Zager PG Johnson ACM Hanson S The mevalonate pathway during acute tubular injury.Am J Pathol. 2002; 161: 681-692Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar The validity of using the contralateral kidneys as controls was ascertained by identifying a lack of cholesterol/cholesterol ester accumulation in them, compared with kidneys obtained from six mice undergoing sham unilateral ischemia surgery. ChIP assays were done using the Matrix ChIP platform in 96-well polystyrene high-binding capacity microplates (no. 9018; Corning, Corning, NY).41Flanagin S Nelson JD Castner DG Denisenko O Bomsztyk K Microplate-based chromatin immunoprecipitation method, Matrix ChIP: a platform to study signaling of complex genomic events.Nucleic Acids Res. 2008; 36: e17Crossref PubMed Scopus (64) Google Scholar The following reagents and equipment were used: protein A (no. P7837; Sigma, St. Louis, MO); proteinase K (no. 25530hyphen]015; Invitrogen, Carlsbad, CA); formaldehyde (no. 2106-02; J.T. Baker, Phillipsburg, NJ); bovine serum albumin (no. A9647, Sigma); phenylmethyl sulfonyl fluoride (no. P-7626, Sigma); leupeptin (no. L-2884, Sigma); SYBR Green PCR master mix (2×SensiMix, no. QT6T3; Quantace, Norwood, MA); salmon sperm DNA (no. D1626, Sigma); Misonix Sonicator 3000 with micro tip (no. S3000; Misonix, Farmingdale, NY); ultrasonic bath (no. B3510-MT CPN-952-316; Branson, Danbury, CT); heat blocks (analog heat block, no. 13259032: VWR Scientific, West Chester, PA; Isotemp 125: Fisher Scientific, Pittsburgh, PA); quantitative PCR (ABI 7900HT system; ABI Biotechnology, Foster City, CA); and MixMate (Eppendorf, Westbury, NY). The following buffers were used: phosphate-buffered saline (PBS): 137 mmol/L NaCl, 10 mmol/L Na phosphate, 2.7 mmol/L KCl, pH 7.4; TE buffer: 10 mmol/L Tris, 1 mmol/L ethylenediaminetetraacetic acid, pH 7.0; immunoprecipitation (IP) buffer: 150 mmol/L NaCl, 50 mmol/L Tris-HCl, pH 7.5, 5 mmol/L ethylenediaminetetraacetic acid, NP-40 (0.5% v/v), Triton X-100 (1.0% v/v); blocking buffer: 5% bovine serum albumin, 100 μg/ml sheared salmon sperm DNA in IP buffer; elution buffer: 25 mmol/L Tris base, 1 mmol/L ethylenediaminetetraacetic acid, pH 9.8, 200 μg/ml proteinase K (20 mg/ml stock, stored at −20°C). Approximately 25 mg of minced renal cortex was fixed with formaldehyde (final concentration 1.42% in PBS for 15 minutes; 22°C) and then quenched with 125 mmol/L glycine (5 minutes, 22°C). The cross-linked tissues were then extensively washed with PBS (4°C). To shear the chromatin, the washed cross-linked tissue pellets were resuspended in 1 ml of IP buffer (containing the following inhibitors: 0.5 mmol/L dithiothreitol, 10 μg/ml leupeptin, 0.5 mmol/L phenylmethyl sulfonyl fluoride, 30 mmol/L p-nitrophenyl phosphate, 10 mmol/L NaF, 0.1 mmol/L Na3VO4, 0.1 mmol/L Na2MoO4, and 10 mmol/L β-glycerophosphate) and sheared using six rounds of sonication (power 5, 15 seconds, on ice). The suspension was cleared by centrifugation at 12,000 × g (10 minutes at 4°C), and the supernatant, representing sheared chromatin, was aliquoted and stored at −80°C. Ninety-six-well plates were washed once with 200 μl of PBS per well and were incubated overnight with 0.2 μg of protein A in 100 μl of PBS per well. After washing (200 μl of PBS per well), well walls were blocked with 200 μl of blocking buffer (15 to 60 minutes, 22°C). The wells were cleared and the used antibodies (Table 1) were added with 100 μl of blocking buffer per well (60 minutes, 22°C). Chromatin samples (5.0-μl chromatin preparations/100 μl of blocking buffer) were added (100 μl/well) and plates were floated in an ultrasonic water bath (60 minutes, 4°C) to accelerate protein-antibody binding.41Flanagin S Nelson JD Castner DG Denisenko O Bomsztyk K Microplate-based chromatin immunoprecipitation method, Matrix ChIP: a platform to study signaling of complex genomic events.Nucleic Acids Res. 2008; 36: e17Crossref PubMed Scopus (64) Google Scholar The wells were washed three times with 200 μl of IP buffer and 1 times with 200 μl of TE buffer. Wells were incubated with 100 μl of elution buffer (15 minutes at 55°C, followed by 15 minutes at 95°C). Total DNA (input) was isolated using the same plate and concurrently with immunoprecipitated DNA by suspending 5.0 μl of chromatin in 100 μl of elution buffer (15 minutes at 55°C, followed by 15 minutes at 95°C). DNA samples were stored (−20°C) in the same Matrix ChIP plates for repeated use.Table 1List of Antibodies Used in Matrix ChIP AssayAntibodyTypeSourceCatalogAmount/ChIPPol II CTD (4h8)MonoclonalGene TexGTX254080.25 μgp65/Rel NF-κBRabbit anti-serumSee Reference 70na0.5 μlc-JunRabbit polyclonalSanta Cruzsc-0440.5 μgc-FosRabbit polyclonalSanta Cruzsc-6520.5 μgSREBP-1MonoclonalPharMingen67351A1.0 μgSREBP-2MonoclonalBD Pharmingen5570371.0 mgHO-1Rabbit polyclonalOncogene ResearchPC3401.0 μgHSP-72/73MonoclonalOncogene ResearchHSP011.0 μgH2A.ZRabbit polyclonalAbcamab41740.5 μgH3K4m3Rabbit polyclonalAbcamAb85800.5 μgH3K9AcRabbit polyclonalCell SignalingNo. 96710.5 μgThese antibodies were used in the ChIP assay, as described in the text. Open table in a new tab These antibodies were used in the ChIP assay, as described in the text. ChIP DNA samples were assayed by qPCR. The reaction mixture contained 2.5 μl of 2× SYBR Green PCR master mix (SensiMix, Quantace), 2.3 μl of DNA template, and 0.2 μl of primers (10 μmol/L) in 5-μl final volume in a 384-well optical reaction plate (Applied Biosystems, Foster City, CA). Amplification (three step, 40 cycles), data acquisition, and analyses were done using the 7900HT real-time PCR system and SDS Enterprise Database (Applied Biosystems). All PCR reactions were run in triplicate. At least four samples were run for each determination. The used qPCR primers are presented in Table 2. PCR calibration curves were generated for each primer pair from a dilution series of total mouse sheared genomic DNA. The PCR primer efficiency curve was fit to cycle threshold (Ct) versus loge (genomic DNA dilutions) using an r-squared best fit. DNA concentration values for each ChIP and input DNA samples were calculated from their respective average Ct values. Final results were expressed as percent input DNA.41Flanagin S Nelson JD Castner DG Denisenko O Bomsztyk K Microplate-based chromatin immunoprecipitation method, Matrix ChIP: a platform to study signaling of complex genomic events.Nucleic Acids Res. 2008; 36: e17Crossref PubMed Scopus (64) Google ScholarTable 2List of Primers Used for qPCR Analyses in ChIP AnalysesHmgcr (ID15357)Right5′-CAGAACCAGAAGAGGCCTTG-3′−5′ (−5000 bp to TSS)Left5′-GACCTCTGGCGGAGACATAC-3′HmgcrRight5′-GGAAGGACTGCGCTTACG-3′Promoter (−100 bp to TSS)Left5′-GTTGTTAGGGAGACCGTTCG-3′HmgcrRight5′-AGGATCCAAGGACTGTGAGG-3′Exon1Left5′-AGGGCACTCATAATTCCAGC-3′HmgcrRight5′-TGTTCAAGGAGCATGCAAAG-3′Exon19Left5′-CTTACCTGTTGTGAACCATGTG-3′HmgcrRight5′-CAGCTCTCCTATCCAATGCC-3′+5′ flanking (+5000 bp to end of gene)Left5′-TTGAAGGACATGCTGCTCAC-3′β-Actin (ID 11461)Right5′-AGGAAGGAAGGCTGGAAGAG-3′Exon 1Left5′-GCTGAGAGGGAAATTGTTCG-3′β-Globin (ID 2440)Right5′-ACCCATGATAGCAGAGGCAG-3′Int 1-Exon 2Left5′-GGTGCTTGGAGACAGAGGTC-3′Primers used in qPCR analyses after chromatin immunoprecipitation. Open table in a new tab Primers used in qPCR analyses after chromatin immunoprecipitation. All values are presented as means ± 1 SEM. Paired Student's t-test was used to compare results obtained from left versus right kidney samples. Comparisons between different mice were performed by unpaired Student's t-test. An n of four to eight tissue samples were used for all comparisons. Significance was judged by a P value of <0.05. We previously demonstrated that by 18 to 24 hours after ischemia-reperfusion (I/R) injury, an ∼25 to 50% increase in renal cortical cholesterol levels result.28Zager RA Burkhart KM Johnson ACM Sacks BM Increased proximal tubular cholesterol content: implications for cell injury and "acquired cytoresistance.".Kidney Int. 1999; 56: 1788-1797Crossref PubMed Scopus (69) Google Scholar The present study demonstrates the durability of this response: at 3 days after unilateral renal ischemia, a time that corresponds to persistent renal tubular
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