Altered renal lipid metabolism and renal lipid accumulation in human diabetic nephropathy

Animal models link ectopic lipid accumulation to renal dysfunction, but whether this process occurs in the human kidney is uncertain. To this end, we investigated whether altered renal TG and cholesterol metabolism results in lipid accumulation in human diabetic nephropathy (DN). Lipid staining and the expression of lipid metabolism genes were studied in kidney biopsies of patients with diagnosed DN (n = 34), and compared with normal kidneys (n = 12). We observed heavy lipid deposition and increased intracellular lipid droplets. Lipid deposition was associated with dysregulation of lipid metabolism genes. Fatty acid β-oxidation pathways including PPAR-α, carnitine palmitoyltransferase 1, acyl-CoA oxidase, and L-FABP were downregulated. Downregulation of renal lipoprotein lipase, which hydrolyzes circulating TGs, was associated with increased expression of angiopoietin-like protein 4. Cholesterol uptake receptor expression, including LDL receptors, oxidized LDL receptors, and acetylated LDL receptors, was significantly increased, while there was downregulation of genes effecting cholesterol efflux, including ABCA1, ABCG1, and apoE. There was a highly significant correlation between glomerular filtration rate, inflammation, and lipid metabolism genes, supporting a possible role of abnormal lipid metabolism in the pathogenesis of DN. These data suggest that renal lipid metabolism may serve as a target for specific therapies aimed at slowing the progression of glomerulosclerosis. Animal models link ectopic lipid accumulation to renal dysfunction, but whether this process occurs in the human kidney is uncertain. To this end, we investigated whether altered renal TG and cholesterol metabolism results in lipid accumulation in human diabetic nephropathy (DN). Lipid staining and the expression of lipid metabolism genes were studied in kidney biopsies of patients with diagnosed DN (n = 34), and compared with normal kidneys (n = 12). We observed heavy lipid deposition and increased intracellular lipid droplets. Lipid deposition was associated with dysregulation of lipid metabolism genes. Fatty acid β-oxidation pathways including PPAR-α, carnitine palmitoyltransferase 1, acyl-CoA oxidase, and L-FABP were downregulated. Downregulation of renal lipoprotein lipase, which hydrolyzes circulating TGs, was associated with increased expression of angiopoietin-like protein 4. Cholesterol uptake receptor expression, including LDL receptors, oxidized LDL receptors, and acetylated LDL receptors, was significantly increased, while there was downregulation of genes effecting cholesterol efflux, including ABCA1, ABCG1, and apoE. There was a highly significant correlation between glomerular filtration rate, inflammation, and lipid metabolism genes, supporting a possible role of abnormal lipid metabolism in the pathogenesis of DN. These data suggest that renal lipid metabolism may serve as a target for specific therapies aimed at slowing the progression of glomerulosclerosis. Diabetic nephropathy (DN) is an increasing cause of morbidity and mortality worldwide and the leading cause of chronic kidney disease (CKD). Dyslipidemia in patients with type 2 diabetes is a reversible risk factor for the progression of kidney disease and cardiovascular mortality (1Rutledge J.C. Ng K.F. Aung H.H. Wilson D.W. Role of triglyceride-rich lipoproteins in diabetic nephropathy.Nat. Rev. Nephrol. 2010; 6: 361-370Crossref PubMed Scopus (99) Google Scholar, 2Cooper M.E. Jandeleit-Dahm K.A. Lipids and diabetic renal disease.Curr. Diab. Rep. 2005; 5: 445-448Crossref PubMed Scopus (22) Google Scholar). Sustained hyperglycemia in diabetes promotes FA synthesis and TG accumulation. Elevated serum TGs, FFAs, and modified cholesterol cause ectopic lipid accumulation in nonadipose tissues, including the pancreas, heart, liver, and blood vessel walls (3Schulze P.C. Myocardial lipid accumulation and lipotoxicity in heart failure.J. Lipid Res. 2009; 50: 2137-2138Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 4Li W.Y. Yao C.X. Zhang S.F. Wang S.L. Wang T.Q. Xiong C.J. Li Y.B. Zang M.X. Improvement of myocardial lipid accumulation and prevention of PGC-1α induction by fenofibrate.Mol. Med. Rep. 2012; 5: 1396-1400PubMed Google Scholar, 5Marfella R. Di Filippo C. Portoghese M. Barbieri M. Ferraraccio F. Siniscalchi M. Cacciapuoti F. Rossi F. D'Amico M. Paolisso G. Myocardial lipid accumulation in patients with pressure-overloaded heart and metabolic syndrome.J. Lipid Res. 2009; 50: 2314-2323Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 6Sharma S. 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Histopathol. 1998; 13: 169-179PubMed Google Scholar), renal lipotoxicity or “fatty kidney” is an unappreciated and not well documented clinical entity, partly due to the confounding multiple coexisting risk factors in DN (14Herman-Edelstein M. Thomas M.C. Thallas-Bonke V. Saleem M. Cooper M.E. Kantharidis P. Dedifferentiation of immortalized human podocytes in response to transforming growth factor-β: a model for diabetic podocytopathy.Diabetes. 2011; 60: 1779-1788Crossref PubMed Scopus (102) Google Scholar). Lipotoxicity and lipid accumulation cause podocyte dysfunction and apoptosis (7Proctor G. Jiang T. Iwahashi M. Wang Z. Li J. Levi M. Regulation of renal fatty acid and cholesterol metabolism, inflammation, and fibrosis in Akita and OVE26 mice with type 1 diabetes.Diabetes. 2006; 55: 2502-2509Crossref PubMed Scopus (223) Google Scholar, 8Kim H.J. Moradi H. Yuan J. Norris K. Vaziri N.D. 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Tonolo G. Role of oxidized low density lipoproteins and free fatty acids in the pathogenesis of glomerulopathy and tubulointerstitial lesions in type 2 diabetes.Nutr. Metab. Cardiovasc. Dis. 2011; 21: 79-85Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 16Wang Z. Jiang T. Li J. Proctor G. McManaman J.L. Lucia S. Chua S. Levi M. Regulation of renal lipid metabolism, lipid accumulation, and glomerulosclerosis in FVBdb/db mice with type 2 diabetes.Diabetes. 2005; 54: 2328-2335Crossref PubMed Scopus (236) Google Scholar, 17Mishra R. Emancipator S.N. Miller C. Kern T. Simonson M.S. Adipose differentiation-related protein and regulators of lipid homeostasis identified by gene expression profiling in the murine db/db diabetic kidney.Am. J. Physiol. Renal Physiol. 2004; 286: F913-F921Crossref PubMed Scopus (75) Google Scholar). The podocyte uptake of oxidized LDL (oxLDL) is mediated mainly by the scavenger chemokine receptor CXCL16 (18Gutwein P. Abdel-Bakky M.S. 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Biochemical and physiological function of stearoyl-CoA desaturase.Am. J. Physiol. Endocrinol. Metab. 2009; 297: E28-E37Crossref PubMed Scopus (461) Google Scholar, 21Dobrzyn P. Sampath H. Dobrzyn A. Miyazaki M. Ntambi J.M. Loss of stearoyl-CoA desaturase 1 inhibits fatty acid oxidation and increases glucose utilization in the heart.Am. J. Physiol. Endocrinol. Metab. 2008; 294: E357-E364Crossref PubMed Scopus (55) Google Scholar). Dysregulation of PPAR activity is a potential cause of metabolic syndrome-related disorders, such as insulin resistance and hyperlipidemia. PPARα and PPARδ regulate expression of genes involved in β-oxidation of FAs. PPARα deficiency has been shown to accelerate dyslipidemia, proteinuria, and renal failure in animal models with DN (16Wang Z. Jiang T. Li J. Proctor G. McManaman J.L. Lucia S. Chua S. Levi M. Regulation of renal lipid metabolism, lipid accumulation, and glomerulosclerosis in FVBdb/db mice with type 2 diabetes.Diabetes. 2005; 54: 2328-2335Crossref PubMed Scopus (236) Google Scholar, 22Kouroumichakis I. Papanas N. Zarogoulidis P. Liakopoulos V. Maltezos E. Mikhailidis D.P. Fibrates: therapeutic potential for diabetic nephropathy?.Eur. J. Intern. Med. 2012; 23: 309-316Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). There is growing evidence that dysregulation of sterol regulatory element binding proteins (SREBPs) contributes to the pathogenesis of DN (23Sun L. Halaihel N. Zhang W. Rogers T. Levi M. Role of sterol regulatory element-binding protein 1 in regulation of renal lipid metabolism and glomerulosclerosis in diabetes mellitus.J. Biol. Chem. 2002; 277: 18919-18927Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). SREBPs serve as the master regulators of cellular FA and cholesterol synthesis; SREBP-1 regulates FA synthesis, whereas SREBP-2 regulates cholesterol synthesis (14Herman-Edelstein M. Thomas M.C. Thallas-Bonke V. Saleem M. Cooper M.E. Kantharidis P. Dedifferentiation of immortalized human podocytes in response to transforming growth factor-β: a model for diabetic podocytopathy.Diabetes. 2011; 60: 1779-1788Crossref PubMed Scopus (102) Google Scholar). In addition, the carbohydrate-responsive element binding protein (ChREBP) regulates the expression of ACC and FAS, and also regulates lipogenesis by induction of the glycolytic enzyme L-pyruvate kinase (L-PK) (24Benhamed F. Denechaud P.D. Lemoine M. Robichon C. Moldes M. Bertrand-Michel J. Ratziu V. Serfaty L. Housset C. Capeau J. et al.The lipogenic transcription factor ChREBP dissociates hepatic steatosis from insulin resistance in mice and humans.J. Clin. Invest. 2012; 122: 2176-2194Crossref PubMed Scopus (281) Google Scholar). Dysregulation of cholesterol metabolism has also been linked to lipotoxicity and lipid accumulation in diabetes. Cholesterol influx into cells is mediated by several independent receptors, including scavenger receptor class A (SR-A1), class B (CD36), lectin-like oxLDL receptor-1 (LOX-1 or OLR-1) (25Urahama Y. Ohsaki Y. Fujita Y. Maruyama S. Yuzawa Y. Matsuo S. Fujimoto T. Lipid droplet-associated proteins protect renal tubular cells from fatty acid-induced apoptosis.Am. J. Pathol. 2008; 173: 1286-1294Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), and LDL receptor (LDLR) (15Nosadini R. Tonolo G. Role of oxidized low density lipoproteins and free fatty acids in the pathogenesis of glomerulopathy and tubulointerstitial lesions in type 2 diabetes.Nutr. Metab. Cardiovasc. Dis. 2011; 21: 79-85Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Cholesterol efflux that prevents cholesterol accumulation is primarily mediated by ATP-binding cassette transporters (ABCA1 and ABCG1). Renal lipoprotein receptor expression correlates with kidney damage (26Lee H.S. Lee S.K. Intraglomerular lipid deposition in renal disease.Miner. Electrolyte Metab. 1993; 19: 144-148PubMed Google Scholar, 27Mayrhofer C. Krieger S. Huttary N. Chang M.W. Grillari J. Allmaier G. Kerjaschki D. Alterations in fatty acid utilization and an impaired antioxidant defense mechanism are early events in podocyte injury: a proteomic analysis.Am. J. Pathol. 2009; 174: 1191-1202Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The aim of this study was to test whether renal dysfunction in human DN is associated with renal lipid accumulation, lipotoxicity, and dysregulation of renal FA and cholesterol metabolism. The study was approved by the Institutional Ethics Committee. Kidney samples were obtained from leftover portions of diagnostic kidney biopsies of patients with DN (n = 34) and normal kidneys (n = 12) from the pathological archives of the Department of Pathology at Rabin Medical Center. All patients had T2D. DN was histologically confirmed by hematoxylin and eosin, periodic acid-schiff, Masson's trichrome staining, immunofluorescence microscopy for immunoglobulin, and complement and electron microscopy (EM) analysis. Criteria for DN included glomerular hypertrophy, diffuse mesangial and focal nodular glomerulosclerosis, arteriolar hyalinosis, focal and segmental glomerulosclerosis, the presence of hyaline drops between Bowman's capsule and epithelial cells, and interstitial fibrosis (28Tervaert T.W. Mooyaart A.L. Amann K. Cohen A.H. Cook H.T. Drachenberg C.B. Ferrario F. Fogo A.B. Haas M. de Heer E. et al.Pathologic classification of diabetic nephropathy.J. Am. Soc. Nephrol. 2010; 21: 556-563Crossref PubMed Scopus (943) Google Scholar). In the DN group, renal biopsy was performed according to clinical indications and in order to exclude the coexistence of other types of kidney disease due the presence of atypical features, including short duration between the diagnosis of diabetes and the onset of nephropathy and/or the absence of concomitant diabetic retinopathy. Cases were defined by the presence of diabetic histological changes consistent with DN and the absence of other potential causes of glomerulonephritis in the pathology evaluation. The degrees of sclerosis, thickening, and fibrosis were evaluated on the basis of an arbitrary scale (0 = normal = 0%, 1 = mild = 20%, 2 = moderate = 20–40%, and 3 = severe = >40%) and were taken from the original pathological evaluations following biopsies. Vascular sclerosis: 0 = normal; 1 = mild; 2 = moderate; 3 = severe. Lumen cells and lumen thrombi: 0 = normal; 1 = increased. Mesangial matrix: 0 = normal; 1 = focal; 2 = increased, mild; 3 = increased, nodular. Bowman's capsule thickening: 0 = normal; 1 = focal; 2 = circumferential. Interstitial fibrosis: 0 = no inflammation; 1 = mild; 2 = severe. Clinical information about the diabetic patients and controls was collected from the patients' files. The glomerular filtration rate (eGFR) was estimated using the CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration) formula (29Levey A.S. Stevens L.A. Schmid C.H. Zhang Y.L. Castro 3rd, A.F. Feldman H.I. Kusek J.W. Eggers P. Van Lente F. Greene T. et al.A new equation to estimate glomerular filtration rate.Ann. Intern. Med. 2009; 150: 604-612Crossref PubMed Scopus (16115) Google Scholar, 30Tachibana H. Ogawa D. Matsushita Y. Bruemmer D. Wada J. Teshigawara S. Eguchi J. Sato-Horiguchi C. Uchida H.A. Shikata K. et al.Activation of liver X receptor inhibits osteopontin and ameliorates diabetic nephropathy.J. Am. Soc. Nephrol. 2012; 23: 1835-1846Crossref PubMed Scopus (41) Google Scholar). Twenty-four hour urine albumin, protein, and creatinine were available to all patients in the last month prior to biopsy. Data on blood pressure were collected from the patients' files on the day of hospitalization for kidney biopsy. As a control group (n = 12), we used diagnostic kidney biopsies from living kidney donors (n = 6) and nonaffected parts of tumor nephrectomy samples (n = 6). Control subjects were defined as having an eGFR >60 (ml/min) and <10% glomerulosclerosis and tubulointerstitial fibrosis. Serum creatinine levels and albuminuria of the patients are presented in Table 1. We grouped the cases according to CKD and rate of eGFR deterioration. Rate of progression was calculated from average rate of eGFR change per month following biopsy until initiation of dialysis or initiation of data collection. All patients were followed up in our department and have all clinical data in our patient files.TABLE 1Clinical and biochemical characteristics of patients with DN and normal controls evaluated in this studyVariableNormal Kidney (n = 12)DN (n = 34)Male sex [n (%)]7 (58)14 (41)Age (years)50.3 ± 21.0bP < 0.001 normal versus DN.58.6 ± 18.3bP < 0.001 normal versus DN.HTN [n (%)]6 (50)33 (97)aP < 0.05 normal versus DN.DM [n (%)]3 (25)18 (100)bP < 0.001 normal versus DN.ACE inhibitor or ARB [n (%)]1 (12.5)24 (70)bP < 0.001 normal versus DN.Statin [n (%)]4 (33)19 (56)aP < 0.05 normal versus DN.Fibrate [n (%)]0 (0)0 (0)BP (mmHg) on admission126/75 ± 26/12154/83 ± 18/11bP < 0.001 normal versus DN.Serum creatinine (mg/dl)0.9 ± 0.32.7 ± 1.8bP < 0.001 normal versus DN.Serum urea (mg/dl)39.2 ± 14.7101.5 ± 56.6aP < 0.05 normal versus DN.Estimated GFR (ml/min/1.73 m2)97.4 ± 29.540.6 ± 30.5bP < 0.001 normal versus DN.Glycated hemoglobin (%)8.7 ± 2.6Plasma LDL-c (mg/dl)115.2 ± 27.2115.8 ± 66.4Plasma HDL-c (mg/dl)51.7 ± 17.149.4 ± 15.9Plasma TGs (mg/dl)154.0 ± 66.4267.6 ± 309.3Serum albumin (mg/dl)4.1 ± 0.53.3 ± 0.6bP < 0.001 normal versus DN.Proteinuria (mg/day) (median)04524.5bP < 0.001 normal versus DN.Weight (kg)62.2 ± 17.585.2 ± 22.1aP < 0.05 normal versus DN.HistologyGlomeruli number29.4 ± 8.924.1 ± 17.3Globally sclerotic glomeruli (%)1.9 ± 3.034.3 ± 27.3bP < 0.001 normal versus DN.Segmentally sclerotic glomeruli (%)0.020.7 ± 23.1bP < 0.001 normal versus DN.Increased mesangial matrix0.3 ± 0.41.9 ± 0.3bP < 0.001 normal versus DN.Bowman's capsule thickening01.9 ± 0.4bP < 0.001 normal versus DN.Tubular atrophy (%)01.7 ± 0.6bP < 0.001 normal versus DN.Interstitial fibrosis (%)01.5 ± 0.6bP < 0.001 normal versus DN.Vascular sclerosis0.1 ± 0.352.2 ± 0.8bP < 0.001 normal versus DN.Interstitial inflammation01.5 ± 0.9bP < 0.001 normal versus DN.a P < 0.05 normal versus DN.b P < 0.001 normal versus DN. Open table in a new tab Kidney biopsy tissue was immediately fixed in Karnovsky's fixative and then 1% buffered osmium tetroxide (method that preserved lipids). The sample was dehydrated in a graded series of ethanol and embedded in an epoxy resin. Tissue was surveyed with a series of 1 μm sections for a representative sample. The selected specimens were thin sectioned, viewed, and photographed with an electron microscope (transmission electron microscope, JEOL 1010) from the Rabin Medical Center pathology department. The sections were read by a nephropathologist to determine kidney morphology and lipid droplets (LDs). 4,4-Difluoro-1,3,5,7,8-pentamethyl-4-Bora-3a,4a-diaza-s-indacene (BODIPY) staining (which stains neutral lipids) was performed on 4 μm-thick sections of fixed frozen kidney DN biopsies and on leftover frozen tissue designated for immunofluorescent microscopy. BODIPY (Molecular Probes, Invitrogen) was diluted in DMSO at a concentration of 1 mg/ml, applied to the kidney section in OCT for 30 min and costained with membranes using a fluorescein-labeled wheat germ agglutinin kit (Molecular Probes, Invitrogen). Oil Red O staining was performed according to the manufacturer's protocol (Sigma) on 6 μm-thick sections of fixed frozen kidney tissue. The kidney sections were rinsed in distilled water, then in 60% isopropanol for 1 min, and stained for 15 min in the Oil Red O working solution (Sigma), then rinsed again for 1 min in 60% isopropanol and returned to distilled water. The slides were counterstained with hematoxylin for 1 min. Filipin staining (which stains cholesterol) (125 μg/ml; Sigma) was performed on 4 μm-thick sections of fixed frozen kidney under light-protected conditions for 2 h at room temperature. Immunofluorescence staining was performed on 4 μm-thick sections of paraffin-embedded human kidney biopsies. We used heat-induced epitope retrieval techniques (antigen-retrieval solution: citrate buffer, pH 6.0; Vector Laboratories). Kidney tissue images were obtained with a Leica TCS SP5 confocal microscope (Tel Aviv University). The adipophilin antibody used was anti-ADFP (AP125) (Fitzgerald). Secondary antibodies were Alexa Fluor 488 tagged. Nuclei were stained with DAPI (Sigma). We used FITC-labeled wheat germ agglutinin (Vector Laboratories) for membrane staining. RNA was extracted from archives of formalin-fixed paraffin-embedded tissue specimens of 34 DN kidney biopsies and 12 normal kidneys. Total RNA was isolated using RNAeasy mini columns (Qiagen, Valencia, CA). The manufacturer's protocol was followed with the exception of increased proteinase K digestion time. RNA quantity and quality were determined by optical density 260:280nm ratio on a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE). RNA was converted to cDNA using RevertAid First Strand cDNA synthesis kit (Fermentas); cDNA was then amplified using TaqMan PreAmp Master Mix (Applied Biosystems) for 14 cycles of preamplification according to the manufacturer's protocol using target gene assays. Candidate lipid and glucose metabolic gene expression were analyzed by real-time RT-PCR, performed as described previously (14Herman-Edelstein M. Thomas M.C. Thallas-Bonke V. Saleem M. Cooper M.E. Kantharidis P. Dedifferentiation of immortalized human podocytes in response to transforming growth factor-β: a model for diabetic podocytopathy.Diabetes. 2011; 60: 1779-1788Crossref PubMed Scopus (102) Google Scholar), using the TaqMan and Syber system based on real-time detection of accumulated fluorescence (ABI Prism Step One; Perkin-Elmer, Foster City, CA). Fluorescence for each cycle was quantitatively analyzed by an ABI Step One sequence detection system (Perkin-Elmer). In order to control for variation in the amount of DNA that was available for PCR in the different samples, gene expression of the target sequence was normalized in relation to the expression of an endogenous control, 18S rRNA or RPLPO (large ribosomal protein). Primer sequences and assay details are available upon request. Values are shown as mean ± SEM unless otherwise specified. Statistical analysis was performed using two-tailed Student's t-test for independent data. Pearson's correlations were calculated using GraphPad software. P < 0.05 was considered significant. RNA samples were prepared from kidney biopsy samples from 34 DN patients and 12 normal controls. The clinical characteristics of the study participants are summarized in Table 1. The DN patients had a higher prevalence of hypertension and obesity. DN patients were more likely to have dyslipidemia and to be on active statin (HMG-CoA reductase inhibitor) therapy. LDL levels were the same in both groups, but diabetics had higher levels of serum TGs. DN patients had decreased eGFR, increased proteinuria, hypoalbuminemia, and increased serum creatinine and urea. Patients in the diabetic group were older and consisted of a higher percentage of women, but these differences did not reach statistical significance. Three control patients were diagnosed with T2D without renal involvement. Histological evaluation showed severe glomerulosclerosis: global sclerosis of 34 ± 17% and segmental sclerosis of 20 ± 23%, tubular atrophy, interstitial fibrosis, vascular sclerosis, mesangial matrix expansion, and marked interstitial inflammation. The phenotype analysis represents all clinical and pathological diversity of DN from early CKD (CKD 1) to advanced CKD (CKD 4–5). Patients with DN showed a progressive decline in kidney function with eGFR deterioration at a rate of 1.0 ± 0.9 ml/min/1.73 m2/month (excluding patients who started dialysis in the 2 months after biopsy). Patients reached dialysis within 29.7 ± 23.6 months after kidney biopsy. As expected in diabetic kidneys (31Gudnason V. Zhou T. Thormar K. Baehring S. Cooper J. Miller G. Humphries S.E. Schuster H. Detection of the low density lipoprotein receptor gene PvuII intron 15 polymorphism using the polymerase chain reaction: association with plasma lipid traits in healthy men and women.Dis. Markers. 1998; 13: 209-220Crossref PubMed Scopus (11) Google Scholar, 32Martini S. Eichinger F. Nair V. Kretzler M. Defining human diabetic nephropathy on the molecular level: integration of transcriptomic profiles with biological knowledge.Rev. Endocr. Metab. Disord. 2008; 9: 267-274Crossref PubMed Scopus (65) Google Scholar), there was increased expression of inflammatory and fibrotic cytokines: TGFβ, TNFα, and collagen. Podocyte marker genes were downregulated. Tubular sodium/glucose cotransporter 2 increased significantly (Fig. 1) with increased expression of the receptor for advanced glycation endproducts (RAGE). EM examination disclosed all the known features of DN, including podocyte process effacement, widening of glomerular basement membrane, and mesangial expansion. We further found extensive accumulation of intracellular LDs in podocyte cells, tubular epithelial cells, and mesangial cells, and also in fenestrated endothelial cells (Fig. 2). LDs are round membrane-coated organelles filled with inert lipids that can be well observed by EM in adipocytes and fatty liver. LDs in DN showed different electron densities depending on the type of lipid and were in different sizes. LDs appeared in clusters mostly seen in podocytes, both in the podocyte cell body and in the major foot processes. Some podocytes were loaded with LDs, while others had no lipid deposits. We studied renal lipid accumulation by using different staining methods on six frozen DN kidney tissue biopsies: Oil Red O, BODIPY staining, and filipin staining (Fig. 3A). We found marked neutral lipid accumulation in both glomeruli and tubulointerstitium, as determined by Oil Red O staining and by BODIPY staining in diabetic kidneys. We also found evidence for cholesterol accumulation by filipin staining (Fig. 3). In the absence of unfixed normal frozen kidney samples and due to the fact that lipids dissolve during routine fixation methods, we used immunostaining of adipophilin (ADRP), a LD-associated protein, to quantify LDs in fixated processed tissue (16Wang Z. Jiang T. Li J. Proctor G. McManaman J.L. Lucia S. Chua S. Levi M. Regulation of renal lipid metabolism, lipid accumulation, and glomerulosclerosis in FVBdb/db mice with type 2 diabetes.Diabetes. 2005; 54: 2328-2335Crossref PubMed Scopus (236) Google Scholar, 17Mishra R. Emancipator S.N. Miller C. Kern T. Simonson M.S. Adipose differentiation-related protein and regulators of lipid