Renal Accumulation of Biglycan and Lipid Retention Accelerates Diabetic Nephropathy
2011; Elsevier BV; Volume: 179; Issue: 3 Linguagem: Inglês
10.1016/j.ajpath.2011.05.016
ISSN1525-2191
AutoresJoel Thompson, Patricia G. Wilson, Katie Brandewie, Deepa Taneja, Liliana Schaefer, Bonnie Mitchell, Lisa R. Tannock,
Tópico(s)Chronic Kidney Disease and Diabetes
ResumoHyperlipidemia worsens diabetic nephropathy, although the mechanism by which renal lipids accumulate is unknown. We previously demonstrated that renal proteoglycans have high low-density lipoprotein (LDL) binding affinity, suggesting that proteoglycan-mediated LDL retention may contribute to renal lipid accumulation. The aim of this study was to determine the relative effect of diabetes and hyperlipidemia on renal proteoglycan content. Diabetic and non-diabetic LDL receptor–deficient mice were fed diets containing 0% or 0.12% cholesterol for 26 weeks, and then kidneys were analyzed for renal lipid and proteoglycan content. Diabetic mice on the high-cholesterol diet had accelerated development of diabetic nephropathy with elevations in urine albumin excretion, glomerular and renal hypertrophy, and mesangial matrix expansion. Renal lipid accumulation was significantly increased by consumption of the 0.12% cholesterol diet, diabetes, and especially by both. The renal proteoglycans biglycan and decorin were detectable in glomeruli, with a significant increase in renal biglycan content in diabetic mice on the high-cholesterol diet. Renal biglycan and renal apolipoprotein B were colocalized, and regression analyses showed a significant relation between renal biglycan and renal apolipoprotein B content. The increased renal biglycan content in diabetic nephropathy probably contributes to renal lipid accumulation and the development of diabetic nephropathy. Hyperlipidemia worsens diabetic nephropathy, although the mechanism by which renal lipids accumulate is unknown. We previously demonstrated that renal proteoglycans have high low-density lipoprotein (LDL) binding affinity, suggesting that proteoglycan-mediated LDL retention may contribute to renal lipid accumulation. The aim of this study was to determine the relative effect of diabetes and hyperlipidemia on renal proteoglycan content. Diabetic and non-diabetic LDL receptor–deficient mice were fed diets containing 0% or 0.12% cholesterol for 26 weeks, and then kidneys were analyzed for renal lipid and proteoglycan content. Diabetic mice on the high-cholesterol diet had accelerated development of diabetic nephropathy with elevations in urine albumin excretion, glomerular and renal hypertrophy, and mesangial matrix expansion. Renal lipid accumulation was significantly increased by consumption of the 0.12% cholesterol diet, diabetes, and especially by both. The renal proteoglycans biglycan and decorin were detectable in glomeruli, with a significant increase in renal biglycan content in diabetic mice on the high-cholesterol diet. Renal biglycan and renal apolipoprotein B were colocalized, and regression analyses showed a significant relation between renal biglycan and renal apolipoprotein B content. The increased renal biglycan content in diabetic nephropathy probably contributes to renal lipid accumulation and the development of diabetic nephropathy. Diabetic nephropathy, the leading cause of end-stage renal disease in the United States, is associated with a dyslipidemia that can exacerbate both the progression of renal disease and the risk of cardiovascular disease. Furthermore, renal disease itself increases the risk of cardiovascular disease, and patients with end-stage renal disease have extremely high mortality rates from cardiovascular disease. Thus, identification of risk factors for and interventions to prevent diabetic nephropathy are of critical public health importance. Several clinical studies have shown that hyperlipidemia aggravates the progression of renal disease, including diabetic nephropathy.1Sato H. Suzuki S. Kobayashi H. Ogino S. Inomata A. Arakawa M. Immunohistological localization of lipoproteins in the glomeruli in renal disease: specifically apoB and apoE.Clin Nephrol. 1991; 36: 127-133PubMed Google Scholar, 2Takemura T. Yoshioka K. Aya N. Murakami K. Matumoto A. Itakura H. Kodama T. Suzuki H. Maki S. lipoproteins and lipoprotein receptors in glomeruli in human kidney diseases.Kidney Int. 1993; 43: 918-927Crossref PubMed Scopus (116) Google Scholar, 3Wheeler D.C. Chana R.S. Interactions between lipoproteins, glomerular cells and matrix.Miner Electrolyte Metab. 1993; 19: 149-164PubMed Google Scholar Glomerular lipid deposition is commonly found on routine biopsies.4Lee H.S. Lee J.S. Koh H.I. Ko K.W. Intraglomerular lipid deposition in routine biopsies.Clin Nephrol. 1991; 36: 67-75PubMed Google Scholar Lipoproteins have relatively free access to the mesangium because of the presence of a fenestrated endothelium without a basement membrane.5Michael A.F. Keane W.F. Raij L. Vernier R.L. Mauer S.M. The glomerular mesangium.Kidney Int. 1980; 17: 141-154Crossref PubMed Scopus (213) Google Scholar Mesangial and glomerular epithelial cells express low-density lipoprotein (LDL) receptors and are capable of endocytosis of bound LDL.6Wasserman J. Santiago A. Rifici V. Holthofer H. Scharschmidt L. Epstein M. Schlondorff D. Interactions of low density lipoprotein with rat mesangial cells.Kidney Int. 1989; 35: 1168-1174Crossref PubMed Scopus (65) Google Scholar, 7Coritsidis G. Rifici V. Gupta S. Rie J. Shan Z.H. Neugarten J. Schlondorff D. Preferential binding of oxidized LDL to rat glomeruli in vivo and cultured mesangial cells in vitro.Kidney Int. 1991; 39: 858-866Crossref PubMed Scopus (105) Google Scholar We and others have demonstrated that hyperlipidemia and diabetes cause accumulation of renal foam cells in animal models,8Sano J. Shirakura S. Oda S. Hara T. Ishihara T. Foam cells generated by a combination of hyperglycemia and hyperlipemia in rats.Pathol Int. 2004; 54: 904-913Crossref PubMed Scopus (15) Google Scholar, 9Spencer M.W. Muhlfeld A.S. Segerer S. Hudkins K.L. Kirk E. LeBoeuf R.C. Alpers C.E. Hyperglycemia and hyperlipidemia act synergistically to induce renal disease in LDL receptor-deficient BALB mice.Am J Nephrol. 2004; 24: 20-31Crossref PubMed Scopus (43) Google Scholar, 10Taneja D. Thompson J. Wilson P. Brandewie K. Schaefer L. Mitchell B. Tannock L.R. Reversibility of renal injury with cholesterol lowering in hyperlipidemic diabetic mice.J Lipid Res. 2010; 51: 1464-1470Crossref PubMed Scopus (14) Google Scholar and we recently demonstrated that lowering of lipid levels via dietary means limits progression of renal injury.10Taneja D. Thompson J. Wilson P. Brandewie K. Schaefer L. Mitchell B. Tannock L.R. Reversibility of renal injury with cholesterol lowering in hyperlipidemic diabetic mice.J Lipid Res. 2010; 51: 1464-1470Crossref PubMed Scopus (14) Google Scholar Increased renal deposition of apolipoproteins (apo) B and/or E is associated with increased mesangial cellularity, increased proteinuria, and increased severity of glomerulosclerosis in a variety of glomerular diseases in humans.1Sato H. Suzuki S. Kobayashi H. Ogino S. Inomata A. Arakawa M. Immunohistological localization of lipoproteins in the glomeruli in renal disease: specifically apoB and apoE.Clin Nephrol. 1991; 36: 127-133PubMed Google Scholar Thus, mesangial accumulation of lipoproteins may exacerbate and accelerate renal injury (reviewed in Wheeler and Chana,3Wheeler D.C. Chana R.S. Interactions between lipoproteins, glomerular cells and matrix.Miner Electrolyte Metab. 1993; 19: 149-164PubMed Google Scholar Kamanna,11Kamanna V.S. Low density lipoproteins and mitogenic signal transduction processes: role in the pathogenesis of renal disease.Histol Histopathol. 2002; 17: 497-505PubMed Google Scholar Abrass,12Abrass C.K. Cellular lipid metabolism and the role of lipids in progressive renal disease.Am J Nephrol. 2004; 24: 46-53Crossref PubMed Scopus (206) Google Scholar and Kamanna et al13Kamanna V.S. Roh D.D. Kirschenbaum M.A. Hyperlipidemia and kidney disease: concepts derived from histopathology and cell biology of the glomerulus.Histol Histopathol. 1998; 13: 169-179PubMed Google Scholar). The mechanisms by which lipid accumulates in the mesangium are unknown. There are numerous commonalities in the pathology of atherosclerosis and nephropathy that suggest common triggers or pathways in the development of these complications. These common features include excess deposition of extracellular matrix, lipid and lipoprotein accumulation, and macrophage infiltration.3Wheeler D.C. Chana R.S. Interactions between lipoproteins, glomerular cells and matrix.Miner Electrolyte Metab. 1993; 19: 149-164PubMed Google Scholar Proteoglycans are a main component of extracellular matrix and participate in the development of atherosclerosis because of their ability to bind and retain lipoproteins.14Williams K.J. Tabas I. The response-to-retention hypothesis of early atherogenesis.Arterioscler Thromb Vasc Biol. 1995; 15: 551-561Crossref PubMed Google Scholar, 15Tabas I. Williams K.J. Boren J. Subendothelial lipoprotein retention as the initiating process in atherosclerosis: update and therapeutic implications.Circulation. 2007; 116: 1832-1844Crossref PubMed Scopus (977) Google Scholar, 16Tannock L.R. King V.L. Proteoglycan mediated lipoprotein retention: a mechanism of diabetic atherosclerosis.Rev Endocr Metab Disord. 2008; 9: 289-300Crossref PubMed Scopus (40) Google Scholar The main proteoglycans synthesized by mesangial cells are the large chondroitin sulfate proteoglycan versican, the small dermatan sulfate proteoglycans biglycan and decorin, and the heparan sulfate proteoglycan perlecan, and earlier studies have shown altered renal proteoglycan synthesis in diabetes.17Hadad S.J. Michelacci Y.M. Schor N. Proteoglycans and glycosaminoglycans synthesized in vitro by mesangial cells from normal and diabetic rats.Biochim Biophys Acta. 1996; 1290: 18-28Crossref PubMed Scopus (28) Google Scholar, 18Silbiger S. Schlondorff D. Crowley S. Rosenberg L. Choi H. Hatcher V. Gordon P. The effect of glucose on proteoglycans produced by cultured mesangial cells.Diabetes. 1993; 42: 1815-1822Crossref PubMed Scopus (20) Google Scholar, 19Ziyadeh F.N. The extracellular matrix in diabetic nephropathy.Am J Kidney Dis. 1993; 22: 736-744PubMed Scopus (231) Google Scholar Previously, we demonstrated that renal proteoglycans exhibit high-affinity binding to LDL, with affinity constants in the plausible physiological range (Kd 14 ± 5 μg/mL LDL).20Tannock L.R. Proteoglycans can mediate renal lipoprotein retention.Diabetologia. 2006; 49: 1115-1116Crossref PubMed Scopus (5) Google Scholar Thus, similar to atherosclerosis, renal lipid accumulation could be mediated, at least in part, via retention by renal proteoglycans. However, it is controversial whether proteoglycans accumulate in the mesangium of diabetic nephropathy.21Schaefer L. Raslik I. Grone H.J. Schonherr E. Macakova K. Ugorcakova J. Budny S. Schaefer R.M. Kresse H. Small proteoglycans in human diabetic nephropathy: discrepancy between glomerular expression and protein accumulation of decorin, biglycan, lumican, and fibromodulin.FASEB J. 2001; 15: 559-561Crossref PubMed Scopus (118) Google Scholar, 22Stokes M.B. Holler S. Cui Y. Hudkins K.L. Eitner F. Fogo A. Alpers C.E. Expression of decorin, biglycan, and collagen type I in human renal fibrosing disease.Kidney Int. 2000; 57: 487-498Crossref PubMed Scopus (83) Google Scholar The aim of this study was to determine the relative effect of diabetes and hypercholesterolemia on renal proteoglycan content during the development of diabetic nephropathy. Hyperlipidemic LDL receptor–deficient (LDLR−/−) mice were selected as the model for this study because we have previously demonstrated that this model develops diabetic nephropathy with overt renal lipid accumulation, which is accelerated in the setting of hypercholesterolemia.10Taneja D. Thompson J. Wilson P. Brandewie K. Schaefer L. Mitchell B. Tannock L.R. Reversibility of renal injury with cholesterol lowering in hyperlipidemic diabetic mice.J Lipid Res. 2010; 51: 1464-1470Crossref PubMed Scopus (14) Google Scholar Chemicals and reagents were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise specified. LDLR−/− mice (C57BL/6J genetic background; generously provided by Alan Daugherty, Lexington, KY) were selected as the model for this study. Unlike many mouse models, LDLR−/− mice carry their cholesterol in LDL particles, develop further elevations in cholesterol when fed high-cholesterol diets, and are susceptible to renal injury. Female mice were used because male mice can develop a type 2 diabetes–like phenotype on high-fat/high-cholesterol diets.23Merat S. Casanada F. Sutphin M. Palinski W. Reaven P.D. Western-type diets induce insulin resistance and hyperinsulinemia in LDL receptor-deficient mice but do not increase aortic atherosclerosis compared with normoinsulinemic mice in which similar plasma cholesterol levels are achieved by a fructose-rich diet.Arterioscler Thromb Vasc Biol. 1999; 19: 1223-1230Crossref PubMed Scopus (128) Google Scholar Mice were housed in a specific pathogen-free facility with 12-hour light/dark cycles and had free access to food and water. These studies were approved by the Animal Care and Use Committees of the University of Kentucky and the Lexington Veterans Affairs Medical Center. Insulin-deficient diabetes was induced with repeated low-dose streptozotocin (STZ). Eight-week-old mice received daily i.p. injections of STZ 40 mg/kg for 5 days and then a second series of injections at the age of 10 weeks. Non-diabetic mice received an identical schedule of injections of the citrate buffer. Hyperglycemia was confirmed at age 11 weeks, then mice were started on diets containing either 0% cholesterol (0% diet; 10.8% calories from fat) or 0.12% cholesterol (0.12% diet; 40% calories from fat; TD000241 and TD000242, respectively; Harlan Teklad, Madison WI) diets at age 12 weeks, as previously described.24Renard C.B. Kramer F. Johansson F. Lamharzi N. Tannock L.R. Herrath M.G. Chait A. Bornfeldt K.E. Diabetes and diabetes-associated lipid abnormalities have distinct effects on initiation and progression of atherosclerotic lesions.J Clin Invest. 2004; 114: 659-668Crossref PubMed Scopus (186) Google Scholar Mice were fed the indicated diets for 26 weeks. All mice were weighed weekly. Blood glucose was measured from the tail vein every 4 weeks, when mice lost weight, or when bedding was excessively wet, which indicated significant hyperglycemia and dehydration (Freestyle Flash Complete Blood Glucose Monitoring System; Abbott Laboratories, Abbott Park, IL). Most diabetic mice received insulin in the form of slow-release subcutaneous pellets (insulin release rate 0.1 U/24 hours per implant for >30 days; Linshin Canada Inc., ON, Canada) to avoid or reverse weight loss, but insulin dose was not titrated to achieve euglycemia (see Supplemental Figure S1A at http://ajp.amjpathol.org). Insulin administration (one pellet at a time) was repeated every 2 to 5 weeks as needed. Systolic blood pressure was measured five times per week in conscious mice via tail cuff apparatus (Visitech Systems Inc., Apex, NC) during weeks 8, 16, and 24 after 1 week of acclimation. The blood pressure was measured by the same operator at the same time each day, and daily measurements within each week were averaged. Mice were bled before receiving STZ or citrate (baseline) and then during weeks 14 and 26. Levels of cholesterol, triglyceride, and glycated hemoglobin were measured as described previously.24Renard C.B. Kramer F. Johansson F. Lamharzi N. Tannock L.R. Herrath M.G. Chait A. Bornfeldt K.E. Diabetes and diabetes-associated lipid abnormalities have distinct effects on initiation and progression of atherosclerotic lesions.J Clin Invest. 2004; 114: 659-668Crossref PubMed Scopus (186) Google Scholar Plasma TGF-β was measured with the TGF-β1 Emax ImmunoAssay System (Promega, Madison, WI) according to the manufacturer's directions. Each mouse was housed individually for 24 hours in metabolic cages during weeks 9, 17, and 25 for collection of urine. Commercially available kits were used to measure urinary albumin (Exocell, Inc., Philadelphia, PA) and urinary creatinine (R&D Systems, Minneapolis, MN), and data are expressed as milligram of albumin per gram of creatinine. After 26 weeks on diet, mice were anesthetized and then perfused at constant, near-physiological pressure through the left ventricle with 10 mL of sterile PBS. The kidneys were removed, decapsulated, and weighed. The right kidney was divided transversely, with one-half embedded in optimum cutting temperature compound and the other half snap frozen in liquid nitrogen. The left kidney was divided transversely, and the halves were fixed in 4% paraformaldehyde then embedded in paraffin. For histologic analyses 4-μm tissue sections were stained with PAS reagent and photographed. Sections were examined by two blinded observers (D.T. and L.R.T.), and matrix accumulation was scored with a semiquantitative scale as previously described.25Taneda S. Pippin J.W. Sage E.H. Hudkins K.L. Takeuchi Y. Couser W.G. Alpers C.E. Amelioration of diabetic nephropathy in SPARC-null mice.J Am Soc Nephrol. 2003; 14: 968-980Crossref PubMed Scopus (86) Google Scholar Glomerular cross-sectional area was measured in ≥30 glomeruli per mouse in glomeruli located in the outer cortex sectioned through the glomerular tuft, using computer-assisted morphometry (Image Pro; Media Cybernetics Inc., Bethesda, MD). Renal disease was evaluated by our expert renal pathologist (B.M.) who was blinded to group. Frozen sections (5-μm thick) of optimum cutting temperature compound were stained with oil red O and photographed. Immunohistochemistry for apoB (antibody recognizes the apoB48 and apoB100; BioDesign, Saco, ME), biglycan, decorin (both R&D Systems), versican (Chemicon, Temecula, CA), and perlecan (Lab Vision-NeoMarkers, Fremont, CA) was performed on 4-μm thick paraffin sections as previously described.26Huang F. Thompson J.C. Wilson P.G. Aung H.H. Rutledge J.C. Tannock L.R. Angiotensin II increases vascular proteoglycan content preceding and contributing to atherosclerosis development.J Lipid Res. 2008; 49: 521-530Crossref PubMed Scopus (52) Google Scholar Intensity of staining within individual glomeruli was quantified with computer-assisted morphometry with the use of ImageJ software version 1.42q (NIH, Bethesda, MD). Renal content of proteoglycan, apoB, and TGF-β (G1221; Promega) was also evaluated by Western blot analyses on total protein extracted from frozen kidneys, as previously described.27Schonherr E. Jarvelainen H.T. Kinsella M.G. Sandell L.J. Wight T.N. Platelet derived growth factor and transforming growth factor-beta1 differentially affect the synthesis of biglycan and decorin by monkey arterial smooth muscle cells.Arterioscler Thromb. 1993; 13: 1026-1036Crossref PubMed Google Scholar Actin was used as the loading control (A2066; Sigma-Aldrich). Blots were scanned, and densitometry was performed with ImageJ software. Data are shown as relative apoB, biglycan, or TGF-β densitometry corrected for actin densitometry. Total renal RNA was isolated with the standard TRIzol method (Invitrogen, Carlsbad, CA), and biglycan expression was evaluated by real-time RT-PCR as previously described28Cai L. Ji A. de Beer F.C. Tannock L.R. van der Westhuyzen D.R. SR-BI protects against endotoxemia in mice through its roles in glucocorticoid production and hepatic clearance.J Clin Invest. 2007; 118: 364-375Crossref Scopus (124) Google Scholar with the use of forward primer 5′-CACCTGGACCACAACAAAA-3′ and reverse primer 5′-TCCGAATCTGATTGTGACCTA-3′. Data are expressed corrected for 18S expression. Colocalization of apoB with proteoglycans was first evaluated by comparing adjacent sections immunostained for apoB and the various proteoglycans. To validate colocalization single sections were double-stained for apoB and biglycan or for apoB and decorin and analyzed by confocal microscopy with the use of a Leica AOBS TCS SP5 inverted laser scanning confocal microscope (Leica Microsystems Inc. Mannheim, Germany). Negative controls were obtained with isotype-matched irrelevant antibodies, no primary antibody, or no secondary antibody. Data are presented as mean ± SEM, unless otherwise described. All data were analyzed by two-way analysis of variance with multiple pairwise comparisons with the use of the Holm-Sidak method (SigmaStat Software Inc., San Jose, CA). P values < 0.05 were considered statistically significant. Mice were made diabetic by STZ injections, whereas control groups received citrate. All mice that received STZ had elevated blood glucose levels by 2 weeks after the injections (see Supplemental Figure S1A at http://ajp.amjpathol.org). Most diabetic mice required insulin periodically to prevent weight loss, but the usage did not differ between groups. The study protocol called for euthanasia of any mouse that lost body weight and did not respond to insulin treatment. One non-diabetic mouse and 7 of 30 diabetic mice died during the study. To investigate the effects of hypercholesterolemia, diabetic and non-diabetic mice were fed diets containing either 0% or 0.12% cholesterol for 26 weeks.24Renard C.B. Kramer F. Johansson F. Lamharzi N. Tannock L.R. Herrath M.G. Chait A. Bornfeldt K.E. Diabetes and diabetes-associated lipid abnormalities have distinct effects on initiation and progression of atherosclerotic lesions.J Clin Invest. 2004; 114: 659-668Crossref PubMed Scopus (186) Google Scholar At the end of the study diabetic mice on either diet had lower body weight than the non-diabetic mice on the same diet (P < 0.001), but consumption of the high-cholesterol diet increased weight in both non-diabetic and diabetic mice (P = 0.002; Table 1; see also Supplemental Figure S1B at http://ajp.amjpathol.org). Induction of diabetes resulted in significantly higher glycated hemoglobin levels compared with non-diabetic mice (P = 0.002; Table 1), and diabetic mice that consumed the 0.12% diet had the highest glycated hemoglobin levels (P < 0.05; Table 1). Blood glucose levels were recorded every 4 weeks throughout the study and were not affected by diet (see Supplemental Figure S1A at http://ajp.amjpathol.org). As expected, consumption of the 0.12% diet resulted in significantly higher total cholesterol levels in both the non-diabetic and diabetic groups (P < 0.001), but there was no effect of diabetes on plasma cholesterol levels (Table 1).24Renard C.B. Kramer F. Johansson F. Lamharzi N. Tannock L.R. Herrath M.G. Chait A. Bornfeldt K.E. Diabetes and diabetes-associated lipid abnormalities have distinct effects on initiation and progression of atherosclerotic lesions.J Clin Invest. 2004; 114: 659-668Crossref PubMed Scopus (186) Google Scholar Furthermore, the groups did not differ in plasma triglyceride levels regardless of diabetes status or diet consumed (Table 1).Table 1Metabolic Characterization0% Diet0.12% DietNon-diabeticDiabeticNon-diabeticDiabeticBody weight (g), study end23.3 ± 1.921.8 ± 1.028.0 ± 2.9⁎P < 0.05 compared with the non-diabetic group on the 0% diet.23.7 ± 0.7†P < 0.05 compared with the non-diabetic group on the 0.12% diet.Glycated hemoglobin (%)7.5 ± 0.48.9 ± 1.1⁎P < 0.05 compared with the non-diabetic group on the 0% diet.7.0 ± 0.211.8 ± 1.0⁎P < 0.05 compared with the non-diabetic group on the 0% diet.†P < 0.05 compared with the non-diabetic group on the 0.12% diet.‡P < 0.05 compared with the diabetic group on the 0% diet.Plasma cholesterol (mg/dL)374 ± 19410 ± 53903 ± 154⁎P < 0.05 compared with the non-diabetic group on the 0% diet.774 ± 51⁎P < 0.05 compared with the non-diabetic group on the 0% diet.‡P < 0.05 compared with the diabetic group on the 0% diet.Triglycerides (mg/dL)118 ± 54143 ± 42103 ± 25251 ± 58Plasma TGF-β (pg/mL)78 ± 782936 ± 803⁎P < 0.05 compared with the non-diabetic group on the 0% diet.1101 ± 3612377 ± 725⁎P < 0.05 compared with the non-diabetic group on the 0% diet.†P < 0.05 compared with the non-diabetic group on the 0.12% diet.Renal weight/ body weight (mg/g)5.0 ± 0.56.0 ± 0.7⁎P < 0.05 compared with the non-diabetic group on the 0% diet.4.6 ± 0.26.2 ± 0.3⁎P < 0.05 compared with the non-diabetic group on the 0% diet.Glomerular cross sectional area (μm2)3954 ± 2813717 ± 2333777 ± 1704946 ± 324⁎P < 0.05 compared with the non-diabetic group on the 0% diet.†P < 0.05 compared with the non-diabetic group on the 0.12% diet.‡P < 0.05 compared with the diabetic group on the 0% diet.Systolic blood pressure (mmHg)120 ± 6120 ± 10112 ± 8114 ± 12Data shown are mean ± SEM for 7 to 14 mice per group as indicated, measured after 26 weeks of the 0% or 0.12% diet and/or diabetes. All analyses were done by two-way analysis of variance with pairwise comparisons by the Holm-Sidak method. P < 0.05 compared with the non-diabetic group on the 0% diet.† P < 0.05 compared with the non-diabetic group on the 0.12% diet.‡ P < 0.05 compared with the diabetic group on the 0% diet. Open table in a new tab Data shown are mean ± SEM for 7 to 14 mice per group as indicated, measured after 26 weeks of the 0% or 0.12% diet and/or diabetes. All analyses were done by two-way analysis of variance with pairwise comparisons by the Holm-Sidak method. Despite the relative resistance of the C57BL6 background strain29Brosius 3rd, F.C. Alpers C.E. Bottinger E.P. Breyer M.D. Coffman T.M. Gurley S.B. Harris R.C. Kakoki M. Kretzler M. Leiter E.H. Levi M. McIndoe R.A. Sharma K. Smithies O. Susztak K. Takahashi N. Takahashi T. Mouse models of diabetic nephropathy.J Am Soc Nephrol. 2009; 20: 2503-2512Crossref PubMed Scopus (430) Google Scholar to the development of diabetic nephropathy, we found a significant elevation in urine albumin excretion in diabetic LDLR−/− mice fed the 0.12% diet (P < 0.05; Figure 1A). Diabetic mice fed either diet developed increased urine albumin excretion compared with the non-diabetic groups fed the same diets as early as 9 weeks of diabetes (not shown). After 26 weeks of diabetes and diets, the diabetic group fed the 0.12% diet had the highest urinary albumin excretion compared with all other groups. Neither diabetes nor diet altered systolic blood pressure at any time (Table 1; showing blood pressure at week 24). Glomerular cross-sectional area was measured in glomeruli sectioned through the tuft, and glomerular mesangial matrix content was estimated with a semiquantitative score. Only diabetic mice fed the 0.12% diet had a significant increase in glomerular area (P < 0.005; Table 1). Diabetic mice had significant mesangial expansion compared with the non-diabetic mice fed the same diets (P < 0.001; Figure 1, B and C). This extent of renal injury is similar to that observed in the endothelial nitric oxide synthase or decorin-deficient models.30Nakagawa T. Sato W. Glushakova O. Heinig M. Clarke T. Campbell-Thompson M. Yuzawa Y. Atkinson M.A. Johnson R.J. Croker B. Diabetic endothelial nitric oxide synthase knockout mice develop advanced diabetic nephropathy.J Am Soc Nephrol. 2007; 18: 539-550Crossref PubMed Scopus (308) Google Scholar, 31Williams K.J. Qiu G. Usui H.K. Dunn S.R. McCue P. Bottinger E. Iozzo R.V. Sharma K. Decorin deficiency enhances progressive nephropathy in diabetic mice.Am J Pathol. 2007; 171: 1441-1450Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar Renal lipid accumulation was evaluated by glomerular staining with the use of the neutral lipid stain oil red O and by immunohistochemistry for apoB. Lipid and lipoprotein accumulation was detectable in glomeruli from diabetic mice fed either diet and from non-diabetic mice fed the 0.12% diet, with the greatest accumulation seen in diabetic mice fed the 0.12% diet (Figure 2, A–C). Minor accumulation of apoB in the interstitium was observed; however, the most intense staining was in the glomeruli (Figure 2B). Western blot analysis of total renal protein similarly showed increased renal apoB content by both diabetes (P = 0.006) and diet (P < 0.001), with the greatest apoB content in diabetic mice fed the 0.12% diet (Figure 2, D and E). The antibody used recognizes both apoB48 and apoB100; however, only apoB100 was seen on Western blot analyses. Glomerular proteoglycan accumulation was evaluated by immunohistochemistry for biglycan, decorin, versican, and perlecan. Biglycan and decorin were the only proteoglycans detectable in any significant amount in any group (Figure 3A), and there was an increase in biglycan staining in glomeruli from diabetic mice fed the 0.12% diet compared with all other groups (Figure 3, B and C). Biglycan showed faint interstitial staining with predominant mesangial and capsular staining. Western blot and densitometry analyses showed increased biglycan content by diabetes and diet; (Figures 3, D and E); however, no significant differences were observed in renal decorin content (Figure 3D) or renal versican content (see Supplemental Figure S2A at http://ajp.amjpathol.org). Biglycan expression measured by real-time PCR was significantly increased by diabetes (P < 0.05; Figure 3F). As expected,32Shankland S.J. Scholey J.W. Ly H. Thai K. Expression of transforming growth factor-beta 1 during diabetic renal hypertrophy.Kidney Int. 1994; 46: 430-442Crossref PubMed Scopus (249) Google Scholar, 33Sharma K. Ziyadeh F.N. Renal hypertrophy is associated with upregulation of TGF-beta 1 gene expression in diabetic BB rat and NOD mouse.Am J Physiol. 1994; 267 (F1094-F1001)PubMed Google Scholar, 34Yamamoto T. Nakamura T. Noble N.A. Ruoslahti E. Border W.A. Expression of transforming growth factor beta is elevated in human and experimental diabetic nephropathy.Proc Natl Acad Sci U S A. 1993; 90: 1814-1818Crossref PubMed Scopus (813) Google Scholar TGF-β levels were significantly higher in the diabetic mice than in the non-diabetic mice (P = 0.005; Table 1). Analysis of renal TGF-β content by Western blot analysis showed a trend toward higher levels in the diabetic mice than in the non-diabetic mice, but it did not reach statistical significance (see Supplemental Figure S2B at h
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