Podocyte Injury–Driven Lipid Peroxidation Accelerates the Infiltration of Glomerular Foam Cells in Focal Segmental Glomerulosclerosis
2015; Elsevier BV; Volume: 185; Issue: 8 Linguagem: Inglês
10.1016/j.ajpath.2015.04.007
ISSN1525-2191
AutoresSatoshi Hara, Namiko Kobayashi, Kazuo Sakamoto, Toshiharu Ueno, Shun Manabe, Yasutoshi Takashima, Juri Hamada, Ira Pastan, Akiyoshi Fukamizu, Taiji Matsusaka, Michio Nagata,
Tópico(s)Ion Transport and Channel Regulation
ResumoIntracapillary foam cell infiltration with podocyte alterations is a characteristic pathology of focal segmental glomerulosclerosis (FSGS). We investigated the possible role of podocyte injury in glomerular macrophage and foam cell infiltration in a podocyte-selective injury model (NEP25 mice) and hypercholesterolemic model [low-density lipoprotein receptor deficiency (LDLR−/−) mice] with doxorubicin–induced nephropathy. Acute podocyte selective injury alone failed to induce glomerular macrophages in the NEP25 mice. However, in the doxorubicin-treated hypercholesterolemic LDLR−/− mice, glomerular macrophages/foam cells significantly increased and were accompanied by lipid deposition and the formation and ingestion of oxidized phospholipids (oxPLs). Glomerular macrophages significantly correlated with the amount of glomerular oxPL. The NEP25/LDLR−/− mice exhibited severe hypercholesterolemia, glomerular lipid deposition, and renal dysfunction. Imaging mass spectrometry revealed that a major component of oxidized low-density lipoprotein, lysophosphatidylcholine 16:0 and 18:0, was present only in the glomeruli of NEP25/LDLR−/− mice. Lysophosphatidylcholine 16:0 stimulated mesangial cells and macrophages, and lysophosphatidylcholine 18:0 stimulated glomerular endothelial cells to express adhesion molecules and chemokines, promoting macrophage adhesion and migration in vitro. In human FSGS, glomerular macrophage–derived foam cells contained oxPLs accompanied by the expression of chemokines in the tuft. In conclusion, glomerular lipid modification represents a novel pathology by podocyte injury, promoting FSGS. Podocyte injury–driven lysophosphatidylcholine de novo accelerated glomerular macrophage–derived foam cell infiltration via lysophosphatidylcholine–mediated expression of adhesion molecules and chemokines in glomerular resident cells. Intracapillary foam cell infiltration with podocyte alterations is a characteristic pathology of focal segmental glomerulosclerosis (FSGS). We investigated the possible role of podocyte injury in glomerular macrophage and foam cell infiltration in a podocyte-selective injury model (NEP25 mice) and hypercholesterolemic model [low-density lipoprotein receptor deficiency (LDLR−/−) mice] with doxorubicin–induced nephropathy. Acute podocyte selective injury alone failed to induce glomerular macrophages in the NEP25 mice. However, in the doxorubicin-treated hypercholesterolemic LDLR−/− mice, glomerular macrophages/foam cells significantly increased and were accompanied by lipid deposition and the formation and ingestion of oxidized phospholipids (oxPLs). Glomerular macrophages significantly correlated with the amount of glomerular oxPL. The NEP25/LDLR−/− mice exhibited severe hypercholesterolemia, glomerular lipid deposition, and renal dysfunction. Imaging mass spectrometry revealed that a major component of oxidized low-density lipoprotein, lysophosphatidylcholine 16:0 and 18:0, was present only in the glomeruli of NEP25/LDLR−/− mice. Lysophosphatidylcholine 16:0 stimulated mesangial cells and macrophages, and lysophosphatidylcholine 18:0 stimulated glomerular endothelial cells to express adhesion molecules and chemokines, promoting macrophage adhesion and migration in vitro. In human FSGS, glomerular macrophage–derived foam cells contained oxPLs accompanied by the expression of chemokines in the tuft. In conclusion, glomerular lipid modification represents a novel pathology by podocyte injury, promoting FSGS. Podocyte injury–driven lysophosphatidylcholine de novo accelerated glomerular macrophage–derived foam cell infiltration via lysophosphatidylcholine–mediated expression of adhesion molecules and chemokines in glomerular resident cells. 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Some studies reported that glomerular foam cells were associated with nephrotic syndrome in humans, including membranous glomerulonephritis, diabetic nephropathy, and IgA glomerulonephritis.16Furuta T. Saito T. Ootaka T. Soma J. Obara K. Abe K. Yoshinaga K. The role of macrophages in diabetic glomerulosclerosis.Am J Kidney Dis. 1993; 21: 480-485Abstract Full Text PDF PubMed Scopus (273) Google Scholar, 17Howie A.J. Changes at the glomerular tip: a feature of membranous nephropathy and other disorders associated with proteinuria.J Pathol. 1986; 150: 13-20Crossref PubMed Scopus (54) Google Scholar, 18Roberts I.S. Pathology of IgA nephropathy.Nat Rev Nephrol. 2014; 10: 445-454Crossref PubMed Scopus (139) Google Scholar However, in these cases, the glomerular foam cells were not always associated with hyperlipidemia. A previous study demonstrated that glomerular foam cells were not correlated with serum cholesterol levels in FSGS patients.7Stokes M.B. Markowitz G.S. D'Agati V.D. 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Our results suggest that podocyte injury promotes hypercholesterolemia-based lipid deposition and specific peroxidation, which activate a molecular network within a glomerular microenvironment that induces macrophage recruitment and foam cell formation in FSGS. NEP25 mice (C57BL/6 background) genetically expressing human CD25 in podocytes were used.3Matsusaka T. Xin J. Niwa S. Niwa S. Kobayashi K. Akatsuka A. Hashizume H. Wang Q.C. Pastan I. Fogo A.B. Ichikawa I. Genetic engineering of glomerular sclerosis in the mouse via control of onset and severity of podocyte-specific injury.J Am Soc Nephrol. 2005; 16: 1013-1023Crossref PubMed Scopus (213) Google Scholar, 4Asano T. Niimura F. Pastan I. Fogo A.B. Ichikawa I. Matsusaka T. Permanent genetic tagging of podocytes: fate of injured podocytes in a mouse model of glomerular sclerosis.J Am Soc Nephrol. 2005; 16: 2257-2262Crossref PubMed Scopus (71) Google Scholar, 25Ueno T. Kobayashi N. Nakayama M. Takashima Y. Ohse T. Pastan I. Pippin J.W. Shankland S.J. Uesugi N. Matsusaka T. Nagata M. Aberrant Notch1-dependent effects on glomerular parietal epithelial cells promotes collapsing focal segmental glomerulosclerosis with progressive podocyte loss.Kidney Int. 2013; 83: 1065-1075Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar In this model, injection of the immunotoxin for human CD25 (LMB2) provokes podocyte-specific injury. Because LMB2 does not damage mouse CD25 but human CD25, LMB does not affect any other organs, including the immune system, except for kidney in mice. Mice aged between 8 and 12 weeks were i.v. injected with LMB2 [4 ng/g body weight (BW) diluted in 100 μL of phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin] or vehicle (VH) through the tail vein. The mice died approximately 2 weeks later because of severe nephrosis. The mice were perfused after 12 days, and kidney tissues were obtained. The LMB2-treated mice (n = 5) were histologically compared with the VH-treated control mice (n = 4). LDLR knockout (LDLR−/−) mice (C57BL/6 background) were used in a hypercholesterolemic mouse model.26Osuga J. Yonemoto M. Yamada N. Shimano H. Yagyu H. Ohashi K. Harada K. Kamei T. Yazaki Y. Ishibashi S. Cholesterol lowering in low density lipoprotein receptor knockout mice overexpressing apolipoprotein E.J Clin Invest. 1998; 102: 386-394Crossref PubMed Scopus (15) Google Scholar Male mice aged between 8 and 12 weeks underwent uninephrectomy of the right kidney under anesthesia using 30% isoflurane (Wako, Osaka, Japan) and were fed a high-fat diet (HFD; 42.6% kcal from fat; Harlan Laboratories, Indianapolis, IN) or a normal diet (ND). Because the genetic background of the LDLR−/− mice revealed low susceptibility to doxorubicin [Adriamycin (ADR); Sigma-Aldrich, St. Louis, MO]–induced nephropathy,27Papeta N. Zheng Z. Schon E.A. Brosel S. Altintas M.M. Nasr S.H. Reiser J. D'Agati V.D. Gharavi A.G. Prkdc participates in mitochondrial genome maintenance and prevents Adriamycin-induced nephropathy in mice.J Clin Invest. 2010; 120: 4055-4064Crossref PubMed Scopus (83) Google Scholar uninephrectomy was performed before the ADR treatment. After 1 week of uninephrectomy, the mice were injected twice with i.v. ADR or VH through the tail vein. ADR was dissolved with ultrapure water and diluted with 0.9% NaCl (final concentration, 2 mg/mL); 0.9% NaCl was used as a VH, with the volume equivalent to the ADR solution. The dose of ADR was 15 mg/kg BW on day 0 and 20 mg/kg BW on day 14. The mice were perfused on day 42, and serum and kidney tissues were obtained. The experimental animal groups were as follows: ADR + ND/LDLR−/− (ADR-treated LDLR−/− fed a ND), ADR + HFD/LDLR−/− (ADR-treated LDLR−/− fed a HFD), and VH + HFD/LDLR−/− (VH-treated LDLR−/− fed a HFD). By crossing the LDLR−/− mice with the NEP25 mice, NEP25/LDLR−/− double-transgenic mice were generated. In this model, the LMB2 injection provokes podocyte-specific injury, together with hypercholesterolemia induced by the HFD. Male mice were initially maintained on an ND. The mice aged between 8 and 12 weeks were then fed a HFD or an ND. After 4 weeks of feeding, 100 μL of LMB2 (4 ng/g BW in PBS containing 0.1% bovine serum albumin) or 100 μL of VH was injected i.v. through the tail vein. The mice were sacrificed after 12 days, and serum and kidney tissues were obtained. On the same day, 24-hour urine was also collected. Kidney tissues were used for histochemical or matrix-assisted laser desorption/deionization time-of-flight IMS (MALDI-TOF-IMS) analysis. We compared the LMB2 + HFD/NEP25/LDLR−/− mice (LMB2-treated NEP25/LDLR−/− mice fed a HFD) with the VH + HFD/LDLR−/− mice (VH-treated LDLR−/− mice fed a HFD) and the LMB2 + ND/NEP25 mice (LMB2-treated NEP25 mice fed an ND). For animal handling and tissue preparation, all experimental procedures were conducted using protocols approved by the Institutional Animal Care and Use Committee of the University of Tsukuba (Tsukuba, Japan; registration numbers 12-380, 13-186, and 14-359). The following biochemical parameters were measured from serum samples: blood urea, nitrogen, creatinine, total cholesterol, triglycerides, and high-density lipoprotein cholesterol using DRI-CHEM7000 (Fujifilm, Tokyo, Japan). In addition, LDL cholesterol was calculated using the Friedrich formula.28Friedewald W.T. Levy R.I. Fredrickson D.S. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge.Clin Chem. 1972; 18: 499-502Crossref PubMed Scopus (64) Google Scholar Urine was analyzed for urinary total protein and creatinine using an automated analyzer (SRL, Tokyo, Japan). Proteinuria was determined by the protein/creatinine ratio. The mice were anesthetized with 30% isoflurane and perfused through the heart with 4% paraformaldehyde (Wako). After kidney dissection, tissue was fixed with 4% paraformaldehyde for paraffin or frozen sections, or with 2% glutaraldehyde for transmission electron microscopy. Paraffin sections (2 μm thick) were processed for periodic acid–Schiff staining, periodic acid–methenamine–silver staining, and immunostaining of human specimens for CD68, oxidized phospholipid (oxPL), macrophage migration inhibitory factor (MIF), lectin-like oxidized LDL receptor-1 (LOX-1), and scavenger receptor class B type I (SR-BI). Frozen sections (5 μm thick) were also processed for oil red O staining and immunostaining of mouse specimens for Wilms tumor protein-1 (WT-1), CD68, and oxPL. The following specific primary antibodies were used: polyclonal rat anti-mouse CD68 antibody (AbD Serotec, Oxford, UK), monoclonal mouse anti-human CD68 antibody (DakoCytomation, Carpinteria, CA), polyclonal goat anti-mouse WT-1 antibody (Santa Cruz Biotechnology, Dallas, TX), monoclonal mouse anti-mouse/human oxPL antibody (Avanti Polar Lipids, Alabaster, AL), polyclonal rabbit anti-mouse/human MIF antibody (Santa Cruz Biotechnology), polyclonal rabbit anti-mouse/human LOX-1 antibody (Abcam, Cambridge, UK), and polyclonal rabbit anti-mouse/human SR-BI antibody (Novus Biologicals, Littleton, CO). For immunostaining, antigen retrieval was performed using a microwave (10 mmol/L citrate buffer; pH 6.0) for WT-1, LOX-1, and SR-BI or proteinase K (Wako) for human CD68 and oxPL. Thereafter, primary antibodies were incubated in a Histofine kit (Nichirei Bioscience, Tokyo, Japan), followed by reaction with peroxidase-conjugated streptavidin (Nichirei Bioscience). Peroxidase activity was visualized using a liquid diaminobenzidine substrate (Dako, Glostrup, Denmark). Hematoxylin was used to stain nuclei. For oil red O staining, 0.3 g of oil red O (Santa Cruz Biotechnology) was diluted with 100 mL of 100% isopropanol (Wako) and incubated overnight. The 0.3% oil red O solution was diluted with ultrapure water to yield 0.05% oil red O solution. After section incubation with 60% isopropanol for 1 minute, the section was stained with 0.05% of oil red O solution for 10 minutes. To measure the oil red O–positive area in the glomeruli, the Lumina Vision imaging software package version 2.4.4 (Mitani Cooperation, Osaka, Japan) was used. The oil red O–positive ratio was calculated by dividing the oil red O–positive area by the glomerular tuft area. The glomerular count was 50 to 100 per animal. Male LMB2 + HFD/NEP25/LDLR−/− (n = 3), VH + HFD/LDLR−/− (n = 4), and LMB2 + ND/NEP25 mice (n = 4) were used for MALDI-TOF-IMS. The animals were anesthetized using 30% isoflurane, and the kidneys were then resected and immediately frozen using dry ice powder. The frozen tissues were sliced into sections (10 μm thick) at −17°C and placed onto 0.1% poly-l-lysine (Sigma-Aldrich)–coated MALDI indium-tin oxide-coated slides (Bruker Daltonics, Bremen, Germany). The sections were dehydrated in a desiccator for 40 minutes and stored at −80°C until use. The matrix solution was subsequently prepared using 10 mg/mL of 2,5-dihydroxybenzoic acid (Wako) in 50% methanol (Wako). Then, 0.2% of trifluoroacetic acid (Wako) was added as an ion-pairing agent. A 2-mL matrix solution was sprayed onto each tissue section using Imageprep (Bruker Daltonics). For the MALDI-TOF-IMS analysis, both profiling and IMS experiments on tissue sections were performed using an Ultraflex MALDI MS instrument (Bruker Daltonics). In the IMS experiments, a 0 to 2000 mass/charge ratio (m/z) region was selected in positive-ionization modes by averaging 500 consecutive laser shots per pixel. The spatial resolution for the imaging data was 80 μm. The laser energy was set at 55% (5.5 μJ), with a repetition rate of 100 Hz. Mass calibration was performed using peptide calibration standard II (Bruker Daltonics) before data acquisition. The raster scan was performed automatically. Software obtained from Bruker Daltonics (flexImaging version 2.1 and flexAnalysis version 3.3) was used for the data analysis. The ion intensities for each m/z value were normalized using flexImaging software, and all spectral intensities were divided by the obtained total ion count value. The ion intensities were evaluated by comparing ion pairs from glomeruli and background normal tissue on the same section after normalization. Candidate lipids were identified using lipid databases, such as LIPIDMAPS (http://www.lipidmaps.org/tools/ms/LMSD_search_mass_options.php, last accessed April 15, 2015). Finally, MS/MS analysis was performed to confirm the chemical structure of the selected lipid of interest using the lipid standards for lysophosphatidylcholine (LPC) 16:0 and 18:0 (both Avanti Polar Lipids). To recognize the location of glomeruli in heat map images of MALDI-TOF-IMS, we used hematoxylin and eosin staining in the same section that was used for measurement of MALDI-TOF-IMS. Briefly, after we performed MALDI-TOF-IMS measurement, we stained the same section for hematoxylin and eosin staining and then recognized the location of glomeruli and depicted as white circles. Finally, we mixed the white circles with heat map images. Our method is a modified version of previous studies.29Lalowski M. Magni F. Mainini V. Monogioudi E. Gotsopoulos A. Soliymani R. Chinello C. Baumann M. 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