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• W2619331588 abstract "Ectopic fat located in the kidney has emerged as a novel cause of obesity-related chronic kidney disease (CKD). In this study, we aimed to investigate whether inflammatory stress promotes ectopic lipid deposition in the kidney and causes renal injury in obese mice and whether the pathological process is mediated by the fatty acid translocase, CD36. High-fat diet (HFD) feeding alone resulted in obesity, hyperlipidemia, and slight renal lipid accumulation in mice, which nevertheless had normal kidney function. HFD-fed mice with chronic inflammation had severe renal steatosis and obvious glomerular and tubular damage, which was accompanied by increased CD36 expression. Interestingly, CD36 deficiency in HFD-fed mice eliminated renal lipid accumulation and pathological changes induced by chronic inflammation. In both human mesangial cells (HMCs) and human kidney 2 (HK2) cells, inflammatory stress increased the efficiency of CD36 protein incorporation into membrane lipid rafts, promoting FFA uptake and intracellular lipid accumulation. Silencing of CD36 in vitro markedly attenuated FFA uptake, lipid accumulation, and cellular stress induced by inflammatory stress. We conclude that inflammatory stress aggravates renal injury by activation of the CD36 pathway, suggesting that this mechanism may operate in obese individuals with chronic inflammation, making them prone to CKD. Ectopic fat located in the kidney has emerged as a novel cause of obesity-related chronic kidney disease (CKD). In this study, we aimed to investigate whether inflammatory stress promotes ectopic lipid deposition in the kidney and causes renal injury in obese mice and whether the pathological process is mediated by the fatty acid translocase, CD36. High-fat diet (HFD) feeding alone resulted in obesity, hyperlipidemia, and slight renal lipid accumulation in mice, which nevertheless had normal kidney function. HFD-fed mice with chronic inflammation had severe renal steatosis and obvious glomerular and tubular damage, which was accompanied by increased CD36 expression. Interestingly, CD36 deficiency in HFD-fed mice eliminated renal lipid accumulation and pathological changes induced by chronic inflammation. In both human mesangial cells (HMCs) and human kidney 2 (HK2) cells, inflammatory stress increased the efficiency of CD36 protein incorporation into membrane lipid rafts, promoting FFA uptake and intracellular lipid accumulation. Silencing of CD36 in vitro markedly attenuated FFA uptake, lipid accumulation, and cellular stress induced by inflammatory stress. We conclude that inflammatory stress aggravates renal injury by activation of the CD36 pathway, suggesting that this mechanism may operate in obese individuals with chronic inflammation, making them prone to CKD. The global increase in chronic kidney disease (CKD) parallels the obesity epidemic, and obese people have an increased risk of progression of CKD (1Wahba I.M. Mak R.H. Obesity and obesity-initiated metabolic syndrome: mechanistic links to chronic kidney disease.Clin. J. Am. Soc. Nephrol. 2007; 2: 550-562Crossref PubMed Scopus (393) Google Scholar). Numerous epidemiologic data suggest that a higher body mass index is a strong independent risk factor for CKD, even after adjusting for traditional risk factors, including blood pressure, diabetes, and dyslipidemia (2Iseki K. Ikemiya Y. Kinjo K. Inoue T. Iseki C. Takishita S. Body mass index and the risk of development of end-stage renal disease in a screened cohort.Kidney Int. 2004; 65: 1870-1876Abstract Full Text Full Text PDF PubMed Scopus (473) Google Scholar, 3Hsu C.Y. McCulloch C.E. Iribarren C. Darbinian J. Go A.S. Body mass index and risk for end-stage renal disease.Ann. Intern. Med. 2006; 144: 21-28Crossref PubMed Scopus (1031) Google Scholar, 4Ejerblad E. Fored C.M. Lindblad P. Fryzek J. McLaughlin J.K. Nyren O. Obesity and risk for chronic renal failure.J. Am. Soc. Nephrol. 2006; 17: 1695-1702Crossref PubMed Scopus (471) Google Scholar). However, it is confusing that many people with congenital and acquired obesity do not suffer renal injury and failure (5St Peter J.V. Hartley G.G. Murakami M.M. Apple F.S. B-type natriuretic peptide (BNP) and N-terminal pro-BNP in obese patients without heart failure: relationship to body mass index and gastric bypass surgery.Clin. Chem. 2006; 52: 680-685Crossref PubMed Scopus (60) Google Scholar, 6McIntyre N. Familial LCAT deficiency and fish-eye disease.J. Inherit. Metab. Dis. 1988; 11: 45-56Crossref PubMed Scopus (42) Google Scholar, 7Noori N. Hosseinpanah F. Nasiri A.A. Azizi F. Comparison of overall obesity and abdominal adiposity in predicting chronic kidney disease incidence among adults.J. Ren. Nutr. 2009; 19: 228-237Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). A large renal biopsy-based clinicopathologic study indicated that a small subset of morbidly obese individuals develops obesity-related glomerulopathy (ORG) (8Kambham N. Markowitz G.S. Valeri A.M. Lin J. D'Agati V.D. Obesity-related glomerulopathy: an emerging epidemic.Kidney Int. 2001; 59: 1498-1509Abstract Full Text Full Text PDF PubMed Scopus (1009) Google Scholar). A study of 257 obese patients in the US revealed only four (1.6%) had dipstick proteinuria, a value much lower than the prevalence of diabetes and hypertension (15% and 23%, respectively) in the same population. These epidemiologic studies implied that CKD does not always occur in obese individuals, which leads to the question: why is CKD not developed by all obese patients? Obesity has been associated with a renal complication that is known as ORG, with predominant histological characteristics of glomerulomegaly and secondary focal segmental glomerulosclerosis (8Kambham N. Markowitz G.S. Valeri A.M. Lin J. D'Agati V.D. Obesity-related glomerulopathy: an emerging epidemic.Kidney Int. 2001; 59: 1498-1509Abstract Full Text Full Text PDF PubMed Scopus (1009) Google Scholar). The mechanisms by which obesity progresses to ORG are not completely understood, but multiple studies have consistently suggested the critical role of glomerular hyperfiltration and insulin resistance (9Tsuboi N. Utsunomiya Y. Hosoya T. Obesity-related glomerulopathy and the nephron complement.Nephrol. Dial. Transplant. 2013; 28: iv108-iv113Crossref PubMed Scopus (32) Google Scholar, 10Praga M. Hernandez E. Morales E. Campos A.P. Valero M.A. Martinez M.A. Leon M. Clinical features and long-term outcome of obesity-associated focal segmental glomerulosclerosis.Nephrol. Dial. Transplant. 2001; 16: 1790-1798Crossref PubMed Scopus (249) Google Scholar, 11Reaven G.M. Banting lecture 1988. Role of insulin resistance in human disease.Diabetes. 1988; 37: 1595-1607Crossref PubMed Scopus (11141) Google Scholar, 12Oterdoom L.H. de Vries A.P. Gansevoort R.T. de Jong P.E. Gans R.O. Bakker S.J. Fasting insulin modifies the relation between age and renal function.Nephrol. Dial. Transplant. 2007; 22: 1587-1592Crossref PubMed Scopus (53) Google Scholar). Gene expression profiles in the glomeruli obtained from ORG patients showed a high expression pattern of genes involved in lipid metabolism [LDL receptor (LDLr), sterol regulatory element-binding protein-1] and inflammation (TNFα, interleukin (IL)-6, and interferon-γ), suggesting that local lipid dysmetabolism and inflammatory processes are also required for the induction and progression of obesity-related CKD (13Carrero J.J. Stenvinkel P. Persistent inflammation as a catalyst for other risk factors in chronic kidney disease: a hypothesis proposal.Clin. J. Am. Soc. Nephrol. 2009; 4: S49-S55Crossref PubMed Scopus (163) Google Scholar, 14Wu Y. Liu Z. Xiang Z. Zeng C. Chen Z. Ma X. Li L. Obesity-related glomerulopathy: insights from gene expression profiles of the glomeruli derived from renal biopsy samples.Endocrinology. 2006; 147: 44-50Crossref PubMed Scopus (129) Google Scholar). Extensive studies suggest that chronic inflammation is a major contributor to progressive renal injury, leading to the development of CKD (15Miyamoto T. Carrero J.J. Stenvinkel P. Inflammation as a risk factor and target for therapy in chronic kidney disease.Curr. Opin. Nephrol. Hypertens. 2011; 20: 662-668Crossref PubMed Scopus (100) Google Scholar). Earlier studies from our laboratory have disclosed the role of inflammatory stress in lipid metabolism disorder. We found that chronic inflammation leads to lipid accumulation in the liver by impairing the balance of lipid influx and efflux (16Ma K.L. Ruan X.Z. Powis S.H. Chen Y. Moorhead J.F. Varghese Z. Inflammatory stress exacerbates lipid accumulation in hepatic cells and fatty livers of apolipoprotein E knockout mice.Hepatology. 2008; 48: 770-781Crossref PubMed Scopus (187) Google Scholar, 17Chen Y. Chen Y. Zhao L. Chen Y. Mei M. Li Q. Huang A. Varghese Z. Moorhead J.F. Ruan X.Z. Inflammatory stress exacerbates hepatic cholesterol accumulation via disrupting cellular cholesterol export.J. Gastroenterol. Hepatol. 2012; 27: 974-984Crossref PubMed Scopus (40) Google Scholar, 18Zhao L. Chen Y. Tang R. Chen Y. Li Q. Gong J. Huang A. Varghese Z. Moorhead J.F. Ruan X.Z. Inflammatory stress exacerbates hepatic cholesterol accumulation via increasing cholesterol uptake and de novo synthesis.J. Gastroenterol. Hepatol. 2011; 26: 875-883Crossref PubMed Scopus (65) Google Scholar). Additionally, chronic inflammation increased lipogenesis in nonadipose tissues and stimulated lipolysis in white adipose tissue, resulting in ectopic lipid deposition in the liver and muscle of mice (19Mei M. Zhao L. Li Q. Chen Y. Huang A. Varghese Z. Moorhead J.F. Zhang S. Powis S.H. Li Q. Inflammatory stress exacerbates ectopic lipid deposition in C57BL/6J mice.Lipids Health Dis. 2011; 10: 110Crossref PubMed Scopus (32) Google Scholar). However, whether the kidney is similarly susceptible to ectopic lipid accumulation as liver or muscle, the contribution of inflammation to ectopic lipid deposition in the kidney, and the progression of obesity-related CKD are largely unknown. Excess accumulation of nonesterified FFA and triglyceride (TG) in the kidney induce cellular lipotoxicity, potentially contributing to CKD development (20Weinberg J.M. Lipotoxicity.Kidney Int. 2006; 70: 1560-1566Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). It is well-known that cells can take up FFA by passive diffusion and also by receptor-mediated mechanisms involving several fatty acid transporters, of which the fatty acid translocase, CD36, is the best characterized (21Wallin T. Ma Z. Ogata H. Jorgensen I.H. Iezzi M. Wang H. Wollheim C.B. Bjorklund A. Facilitation of fatty acid uptake by CD36 in insulin-producing cells reduces fatty-acid-induced insulin secretion and glucose regulation of fatty acid oxidation.Biochim. Biophys. Acta. 2010; 1801: 191-197Crossref PubMed Scopus (35) Google Scholar). CD36 is an integral transmembrane glycoprotein expressed in various tissues, where it is involved in high-affinity uptake of long-chain fatty acid (LCFA) (22Febbraio M. Hajjar D.P. Silverstein R.L. CD36: a class B scavenger receptor involved in angiogenesis, atherosclerosis, inflammation, and lipid metabolism.J. Clin. Invest. 2001; 108: 785-791Crossref PubMed Scopus (927) Google Scholar). Increased CD36 expression was observed in the kidneys of patients with CKD (23Baines R.J. Brunskill N.J. Tubular toxicity of proteinuria.Nat. Rev. Nephrol. 2011; 7: 177-180Crossref PubMed Scopus (77) Google Scholar, 24Hua W. Huang H.Z. Tan L.T. Wan J.M. Gui H.B. Zhao L. Ruan X.Z. Chen X.M. Du X.G. CD36 mediated fatty acid-induced podocyte apoptosis via oxidative stress.PLoS One. 2015; 10: e0127507Crossref PubMed Scopus (85) Google Scholar), suggesting that CD36 plays an important role in the pathogenesis of renal diseases (24Hua W. Huang H.Z. Tan L.T. Wan J.M. Gui H.B. Zhao L. Ruan X.Z. Chen X.M. Du X.G. CD36 mediated fatty acid-induced podocyte apoptosis via oxidative stress.PLoS One. 2015; 10: e0127507Crossref PubMed Scopus (85) Google Scholar, 25Susztak K. Ciccone E. McCue P. Sharma K. Böttinger E.P. Multiple metabolic hits converge on CD36 as novel mediator of tubular epithelial apoptosis in diabetic nephropathy.PLoS Med. 2005; 2: e45Crossref PubMed Scopus (162) Google Scholar, 26Okamura D.M. Pennathur S. Pasichnyk K. López-Guisa J.M. Collins S. Febbraio M. Heinecke J. Eddy A.A. CD36 regulates oxidative stress and inflammation in hypercholesterolemic CKD.J. Am. Soc. Nephrol. 2009; 20: 495-505Crossref PubMed Scopus (108) Google Scholar). In this study, we aimed to determine whether inflammation induces ectopic lipid deposition in the kidney and confers susceptibility to the development of renal disease in high-fat diet (HFD)-fed obese mice and whether CD36-regulated fatty acid uptake is involved in this process. In the casein injection-induced model of chronic inflammation, we found that casein injection in HFD-fed mice led to renal alterations and accompanying systemic abnormalities compatible to human obesity-related CKD, including albuminuria, renal lipid accumulation, and glomerular lesions. These abnormalities were prevented by the loss of CD36 in mice, suggesting that the CD36 pathway contributes to the development of obesity-related CKD under chronic inflammation. Animal care and experimental procedures were performed with approval from the animal care committees of Chongqing Medical University. CD36 knockout (CD36−/−) mice created on a C57BL/6J background were kindly provided by Dr. Maria Febbraio (Lerner Research Institute). Six-week-old male C57BL/6J (WT) mice and CD36−/− mice were used in this study. In one experiment, WT mice were randomly assigned to receive a normal chow diet (NCD) (D12102C; Research Diets) or NCD plus subcutaneous casein injection of 0.5 ml 10% casein every other day or a HFD (60% kcal in fat, D12492; Research Diets) or HFD plus casein injection (HFD+casein) (n = 5 mice per group). In another experiment, WT and CD36−/− mice were fed a HFD plus casein injection (n = 5 mice per group). Mice were euthanized after 10 weeks, and blood and kidney samples were collected for further assessments. Human mesangial cells (HMCs) were cultured in RPMI-1640 growth medium containing 10% fetal bovine serum, 1% insulin-transferrin-sodium selenite, 100 U/ml penicillin, and 100 μg/ml streptomycin. Human kidney 2 (HK2) cells were cultured in RPMI-1640 growth medium containing 10% fetal bovine serum, 100 U/ml penicillin, and 100 mg/ml streptomycin. Experimental medium was prepared with serum-free RPMI-1640 growth medium containing 0.2% fatty acid-free BSA (Sigma, Poole, Dorset, UK), and the cells were subjected to palmitic acid (PA) (0.04 mmol/l) loading in the absence or presence of TNFα (25 ng/ml) or IL-6 (20 ng/ml) for 24 h. PA was obtained from Sigma, and TNFα and IL-6 were purchased from Peprotech (Peprotech Asia, Rehovot, Israel). The serum levels of serum amyloid A (SAA) protein and TNFα were measured using commercial kits (United States Biological, Swampscott, MA) according to the manufacturer's instructions. Serum concentrations of FFA, TG, creatinine, albumin, and blood urea nitrogen (BuN) were determined using an AU2700 automatic biochemical analyzer (Olympus, Tokyo, Japan). Quantitative measurement of FFA and TG levels in cells and kidneys were performed using an enzyme-linked immunosorbent assay (Cusabio Biotech Co. Ltd, Wuhan, China). Renal samples were collected and fixed in 10% formalin. The samples were then embedded in paraffin, sliced to 8 μm in thickness, and stained with hematoxylin-eosin (HE) and periodic acid-silver methenamine (PASM) to evaluate the renal structural changes. For lipid analysis, frozen sections of kidneys were fixed and stained with Oil Red O for 15–30 min, and samples, after being washed, were then stained with hematoxylin for another 2 min. All images were captured using a Zeiss microscope (Zeiss, Jena, Germany). Immunohistochemical studies were performed on sections of formalin-fixed and paraffin-embedded kidney tissues. Endogenous peroxidases were inactivated using 3% H2O2, followed by blocking with goat serum. Sections were incubated overnight (4°C) with anti-collagen 4 (1:200; Santa Cruz, CA), anti-GRP78 (1:200; Bioss, Beijing, China), or anti-IRE1 (1:200; Bioss). Then, the sections were washed and incubated for 45 min with secondary antibody. Histochemical reactions were performed using a diaminobenzidine kit, and sections were counterstained with hematoxylin. All images were captured using a Zeiss microscope (Zeiss). HK2 cells and HMCs cultured on coverslips were washed three times with PBS and fixed with 4% paraformaldehyde for 15 min. Then, cells were permeabilized with 0.25% Triton X-100 and blocked with 1% BSA for 30 min. Incubation with primary antibody against CD36 (1:2,000; Novus) was performed overnight at 4°C, followed by incubation with secondary antibody (1:200; Zhongshan Golden Bridge Biotech, Beijing, China). The cells were washed and stained with DAPI (1:50, Beyotime, Beijing, China) for 1 min. Fluorescence images were obtained using a florescence microscope (Zeiss). To assess the uptake and accumulation of FFA, HMCs and HK2 cells were washed with PBS and loaded with fluorescent probe, BODIPY FL C16 (D3821; Invitrogen, Eugene, OR). The uptake and accumulation of FFA in HMCs and HK2 cells were examined simultaneously in vitro using a confocal microscope until a plateau was obtained, and the fluorescence was measured at an excitation wavelength of 505 nm and an emission wavelength of 510 nm. Total RNA was extracted from white adipose tissue by RNAiso Plus reagent (Takara, Dalian, China). Then, cDNA synthesis and quantitative real-time PCR were performed with commercial kits (Takara) using the Bio-Rad CFX Connect TM real-time system (Bio-Rad, Hercules, CA) according to the manufacturer's instructions. Relative expression compared with that of β-actin was calculated using the comparative cycle threshold method. The total proteins were extracted using RIPA lysis buffer. Sample proteins were separated by SDS-PAGE in a Bio-Rad Mini protean apparatus (Bio-Rad) and transferred to a PVDF membrane (Millipore, Billerica, MA). The membranes were then blocked and incubated with primary antibodies against CD36 (1:200; Novus) followed by incubation with a horseradish peroxidase-labeled secondary antibody (Santa Cruz). Finally, detection procedures were performed using an ECL Advance Western blotting detection kit (Bio-Rad). Plasma membrane lipid rafts, identified as detergent-resistant membranes (DRMs), were prepared by flotation on sucrose density gradients by ultracentrifugation of Triton X-100 lysates, as previously described (27Zeaiter Z. Cohen D. Müsch A. Bagnoli F. Covacci A. Stein M. Analysis of detergent-resistant membranes of Helicobacter pylori infected gastric adenocarcinoma cells reveals a role for MARK2/Par1b in CagA-mediated disruption of cellular polarity.Cell. Microbiol. 2008; 10: 781-794Crossref PubMed Scopus (81) Google Scholar). Briefly, cells were lysed for 20 min on ice in TNE buffer containing 1% Triton X-100 and protease inhibitors. Postnuclear supernatants were added with an equal volume of 80% sucrose, overlaid with 30% sucrose, topped by 5% sucrose, and then run on discontinuous sucrose gradients (40–5%) at 2,260 g for 15 h at 4°C. Fractions divided into 12 parts were collected from the top of the gradient, and equal protein amounts from each fraction were analyzed by immunoblotting. All data are expressed as the mean ± SEM. Statistical analysis was performed using a Student's t-test when only two value sets were compared and one-way ANOVA followed by Tukey's multiple comparison test when the data involved three or more groups. P < 0.05 was considered a statistically significant difference. Subcutaneous casein injection in C57BL/6J mice was previously reported to induce chronic inflammation in vivo (28Wu Y. Wu T. Wu J. Zhao L. Li Q. Varghese Z. Moorhead J.F. Powis S.H. Chen Y. Ruan X.Z. Chronic inflammation exacerbates glucose metabolism disorders in C57BL/6J mice fed with high-fat diet.J. Endocrinol. 2013; 219: 195-204Crossref PubMed Scopus (44) Google Scholar, 29Xu Z.E. Chen Y. Huang A. Varghese Z. Moorhead J.F. Yan F. Powis S.H. Li Q. Ruan X.Z. Inflammatory stress exacerbates lipid-mediated renal injury in ApoE/CD36/SRA triple knockout mice.Am. J. Physiol. Renal Physiol. 2011; 301: F713-F722Crossref PubMed Scopus (21) Google Scholar). In this study, casein injection in NCD-fed mice caused systemic inflammation, revealed by significant increases of TNFα and SAA in serum (Table 1). Injection of casein alone had no effect on body weight and serum lipids (Table 1). Serum BuN concentrations and 24 h urinary albumin excretion were similar between the NCD group and the NCD+casein group; only serum creatinine concentrations were increased in NCD+casein mice (Table 1).TABLE 1Characteristics of mice after the 10 week experimental periodNCDNCD+CaseinHFDHFD+CaseinBody weight (g)21.85 ± 1.0021.41 ± 0.4025.31 ± 2.12aP < 0.05 versus the NCD group.25.51 ± 1.16Serum SAA (ng/ml)19.35 ± 0.8183.26 ± 15.3aP < 0.05 versus the NCD group.17.14 ± 5.686.6 ± 18.28bP < 0.05 versus the HFD group.Serum TNFα (pg/ml)4.39 ± 0.449.55 ± 0.84aP < 0.05 versus the NCD group.2.49 ± 0.1711.43 ± 0.86bP < 0.05 versus the HFD group.Serum FFA (mmol/l)0.78 ± 0.051.00 ± 0.041.54 ± 0.12aP < 0.05 versus the NCD group.1.15 ± 0.08Serum TG (mmol/l)0.99 ± 0.051.05 ± 0.091.41 ± 0.04aP < 0.05 versus the NCD group.1.01 ± 0.01bP < 0.05 versus the HFD group.Serum BuN (mmol/l)7.93 ± 0.189.02 ± 0.168.77 ± 0.2911.90 ± 0.59bP < 0.05 versus the HFD group.Serum Creatinine (μmol/l)4.86 ± 0.417.23 ± 0.22aP < 0.05 versus the NCD group.5.84 ± 0.438.64 ± 0.59bP < 0.05 versus the HFD group.Urinary albumin excretion (μg/day)9.14 ± 0.627.66 ± 0.458.12 ± 0.4712.5 ± 0.48bP < 0.05 versus the HFD group.Mice were fed a NCD, NCD plus casein injection (NCD+Casein), HFD, or HFD plus casein injection (HFD+Casein) for 10 weeks. Values are expressed as mean ± SEM, n = 5 per group.a P < 0.05 versus the NCD group.b P < 0.05 versus the HFD group. Open table in a new tab Mice were fed a NCD, NCD plus casein injection (NCD+Casein), HFD, or HFD plus casein injection (HFD+Casein) for 10 weeks. Values are expressed as mean ± SEM, n = 5 per group. Mice on a HFD exhibited features of obesity and hyperlipidemia, and maintained normal levels of serum cytokines (Table 1). There was no significant change in any of the renal parameters measured in HFD-fed mice compared with NCD-fed mice (Table 1). However, HFD-fed mice that received casein injection had higher concentrations of BuN and creatinine in the serum, and showed significantly increased urinary albumin excretion (Table 1). In addition, casein injection in HFD-fed mice resulted in higher serum cytokine levels and lower serum TG levels (Table 1). These data suggest that the combination of HFD feeding and casein injection aggravates renal injury in mice. The protein expression of TNFα, IL-6, and monocyte chemotactic protein-1 (MCP-1) in the kidney of HFD+casein mice was significantly higher than in HFD-fed mice (supplemental Fig. S1A, B). Oil Red O staining and TG/FFA quantitation revealed much more lipid accumulation in the glomeruli and tubules of the kidneys of HFD+casein mice than those of HFD mice (Fig. 1A, B), suggesting that serum lipids may relocate to the kidneys of obese mice under chronic inflammation. In addition, casein injection in NCD-fed mice did not change renal lipid contents (Fig. 1A, B). Renal histology analysis revealed no obvious morphological change in the mice treated with HFD or casein alone by comparison with the normal NCD-fed mice (Fig. 1C, D), whereas kidney sections from the HFD+casein mice showed glomerulomegaly, disruption of glomerular architecture, and vacuolar degeneration of tubular epithelial cells (Fig. 1C, D). PASM staining showed thickened glomerular basement membrane and increased extracellular matrix in the glomeruli of HFD+casein mice (Fig. 1E). Kidney sections of HFD+casein mice also displayed increased major extracellular matrix protein collagen 4 staining in the glomeruli (Fig. 1E). Additionally, the mRNA expression of Col1α1 and Col4α1 procollagen was increased in HFD+casein mice compared with HFD-fed mice (Fig. 1F). These data, taken together, suggested that chronic inflammation induced by casein initiates the development of obesity-related nephropathy. The expression of CD36 was significantly increased in the kidney of HFD+casein mice compared with the HFD mice (Fig. 2A). Interestingly, CD36−/− mice were protected from renal dysfunction induced by chronic inflammation, as evidenced by normalized serum parameters (urea nitrogen, creatinine, and albumin) and, especially, decreased 24 h urinary albumin excretion (Fig. 2B). CD36 deficiency in mice eliminated inflammation-induced renal lipid accumulation, which was revealed by Oil Red O staining and the quantitative analysis of TG and FFA levels (Fig. 2C, D). Additionally, CD36 deficiency ameliorated renal pathological changes induced by chronic inflammation, caused a decreasing trend in glomerular size, eliminated vacuolar degeneration of tubular epithelial cells, reduced mesangial expansion, and decreased extracellular matrix content (Fig. 2E–H). These data demonstrated that deficiency of CD36 in mice prevents the development of renal injury induced by inflammation. The total protein levels of CD36 detected by Western blot were increased by inflammatory cytokine treatment in both HMCs and HK2 cells (Fig. 3A). By using immunofluorescence, we found a membrane localization of CD36 in HMCs and HK2 cells treated with inflammatory cytokines (Fig. 3B). Then, the effects of inflammatory cytokines on CD36 distribution in lipid rafts, a key membrane microdomain determining CD36 functions (30Eyre N.S. Cleland L.G. Tandon N.N. Mayrhofer G. Importance of the carboxyl terminus of FAT/CD36 for plasma membrane localization and function in long-chain fatty acid uptake.J. Lipid Res. 2007; 48: 528-542Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), were detected by DRM analysis. The CD36 protein in the TNFα-treated groups was highly enriched in the DRM fractions, marked by caveolin, a lipid raft marker. In contrast, the CD36 protein in the control group was less evident within the DRM fractions and showed higher amounts within the non-DRM fractions (Fig. 3C). These data suggest that inflammatory stress not only increases CD36 expression, but also increases its distribution in membrane rafts. It has been demonstrated that CD36-mediated LCFA uptake requires its localization to plasma lipid rafts (30Eyre N.S. Cleland L.G. Tandon N.N. Mayrhofer G. Importance of the carboxyl terminus of FAT/CD36 for plasma membrane localization and function in long-chain fatty acid uptake.J. Lipid Res. 2007; 48: 528-542Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). We determined the effects of inflammatory stress on the cellular uptake for LCFA. Cells were loaded with a fluorescent fatty acid analog BODIPY-C16, and the uptake rate of fluorescent LCFA was determined using fluorescent microscopy. In both HMCs and HK2 cells, TNFα and IL-6 treatment accelerated the efficiency of fluorescent LCFA uptake into cells (Fig. 4A). The quantitative analysis of cellular TG and FFA revealed increased lipid accumulation in HMCs and HK2 cells after inflammatory cytokine treatment (Fig. 4B). These results suggest that inflammatory stress may aggravate lipid accumulation by accelerating fatty acid uptake. To examine the direct role of CD36 in inflammatory stress-mediated lipid accumulation, we silenced the expression of CD36 by transient siRNA transfection. As shown in supplemental Fig. S2A, B, CD36 expression was reduced nearly 4-fold by CD36-siRNA in HMCs and HK2 cells. In the control group, TNFα and IL-6 treatment accelerated fluorescent LCFA uptake in HMCs and HK2 cells, however, the CD36 knockdown largely abrogated the effects of inflammatory cytokines on LCFA uptake (Fig. 5A, B). Additionally, CD36 silence markedly attenuated intracellular TG and FFA accumulation induced by inflammatory cytokines (Fig. 5C). Lipid accumulation has direct toxic effects on renal cells by initiating endoplasmic reticulum (ER) stress and oxidative stress (31Ruan X.Z. Varghese Z. Moorhead J.F. An update on the lipid nephrotoxicity hypothesis.Nat. Rev. Nephrol. 2009; 5: 713-721Crossref PubMed Scopus (211) Google Scholar). ER stress and oxidative stress were enhanced by either TNFα or IL-6 treatment in both HMCs and HK2 cells, as demonstrated by increased mRNA expression of ER stress-related genes, Grp78 and Ire1, and increased production of ROS (Fig. 6A, B). However, when CD36 was silenced in those cells, inflammatory cytokines failed to induce ER stress or oxidative stress (Fig. 6A, B). Similarly, in vivo quantification of GRP78 and IRE-1 protein and gene expression by immunohistochemistry and RT-PCR revealed the increased ER stress in the kidney of HFD+casein mice compared with the HFD mice (Fig. 6C, D). The production of H2O2 was also increased by casein injection in HFD-fed mice compared with HFD-fed mice (Fig. 6E). Additionally, ER stress and oxidative stress were significantly decreased in the kidneys of CD36−/− mice compared with WT mice (Fig. 6F–H). These data suggested that inflammatory stress may trigger lipotoxicity via the CD36-dependent pathway. The purpose of our current study was to clarify the role of inflammatory stress in obesity-induced renal disease. Here, we show that: 1) inflammatory stress induces renal lipid accumulation by promoting fatty acid uptake, as well as renal injury and systemic alterations; and 2) these abnormalities under inflammation are ameliorated in CD36−/− mice. Feeding a HFD to mice is known to induce various systemic alterations, including obesity, hyperglycemia, and abnormal lipi" @default.
• W2619331588 created "2017-06-05" @default.
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• W2619331588 date "2017-07-01" @default.
• W2619331588 modified "2023-10-17" @default.
• W2619331588 title "Inflammatory stress promotes the development of obesity-related chronic kidney disease via CD36 in mice" @default.
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