research article short-term high fat intake does not...

Research ArticleShort-Term High Fat Intake Does Not SignificantlyAlter Markers of Renal Function or Inflammation inYoung Male Sprague-Dawley Rats

Catherine Crinigan Matthew Calhoun and Karen L Sweazea

School of Nutrition and Health Promotion School of Life Sciences Arizona State UniversityTempe AZ 85287 USA

Correspondence should be addressed to Karen L Sweazea karensweazeaasuedu

Received 3 March 2015 Revised 8 June 2015 Accepted 9 June 2015

Academic Editor Jonathan M Hodgson

Copyright copy 2015 Catherine Crinigan et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Chronic high fat feeding is correlated with diabetes and kidney disease However the impact of short-term high fat diets(HFD) is not well-understood Six weeks of HFD result in indices of metabolic syndrome (increased adiposity hyperglycemiahyperinsulinemia hyperlipidemia hyperleptinemia and impaired endothelium-dependent vasodilation) compared to rats fedon standard chow The hypothesis was that short-term HFD would induce early signs of renal disease Young male Sprague-Dawley rats were fed either HFD (60 fat) or standard chow (5 fat) for six weeks Morphology was determined by measuringchanges in renal mass and microstructure Kidney function was measured by analyzing urinary protein creatinine and hydrogenperoxide (H

2O2) concentrations as well as plasma cystatin C concentrations Renal damage was measured through assessment

of urinary oxDNARNA concentrations as well as renal lipid peroxidation tumor necrosis factor alpha (TNF120572) and interleukin6 (IL-6) Despite HFD significantly increasing adiposity and renal mass there was no evidence of early stage kidney disease asmeasured by changes in urinary and plasma biomarkers as well as histology These findings suggest that moderate hyperglycemiaand inflammation produced by short-termHFD are not sufficient to damage kidneys or that the ketogenicHFDmay have protectiveeffects within the kidneys

1 Introduction

Diabetes is currently the leading cause of 44 of newly diag-nosed cases of renal failure [1] as sustained hyperglycemiadamages the filtration membranes of the kidneys resultingin albuminuria and proteinuria In fact proteinuria is oftenused as a surrogate marker for renal disease [2ndash4] As theincidence of diabetes continues to increase throughout theworld rates of albuminuria and diabetic kidney disease(DKD) are likewise expected to increase [2]

The mechanisms leading to the development of chronickidney disease (CKD) in subjects with metabolic syndromehowever are not as well-understood [5] According tothe World Health Organization the criteria for diagnos-ing metabolic syndrome indicate that an individual musthave insulin resistance type 2 diabetes impaired glucose

tolerance or impaired fasting glucose along with two addi-tional criteria abdominal or overall obesity dyslipidemiahypertension or microalbuminuria [6]These characteristicsof metabolic syndrome also increase the risk of developingcardiovascular disease and diabetes which subsequentlyincrease the risk of renal dysfunction and failure [6] Fosteret al [7] examined data from the Framingham Heart Studythat explored the relationship between renal fat accumu-lation hypertension and CKD Participants were assessedfor renal fat by computed tomography Positive associationswere identified between fatty kidney and hypertension andbetween fatty kidney and CKD After adjusting for visceraladipose tissue no association was found between fat infiltra-tion in the kidney and diabetesThese data indicate that theremay be an independent association between hypertensionrenal fat accumulation and CKD [7]

Hindawi Publishing CorporationJournal of Nutrition and MetabolismVolume 2015 Article ID 157520 9 pageshttpdxdoiorg1011552015157520

2 Journal of Nutrition and Metabolism

Deji et al [5] showed that feeding 6-week-oldmice a highfat diet (60 fat) for 12 weeks increased body mass plasmaglucose insulin and triglyceride concentrations and inducedhypertension and kidney disease as evidenced by increasedalbuminuria alterations in renal morphology and renal lipidaccumulation Similarly Altunkaynak et al [8] demonstratedthat feeding adult female Sprague-Dawley rats a moderate fatdiet (30 fat) for 12 weeks caused the animals to becomeoverweight and to develop increased kidney masses andvolumes with significant morphological changes indicativeof renal disease Moreover renal fat accumulation is evidentwith even moderate fat intake in rabbits (10 corn oil +5 lard for 8ndash12 weeks) [9] Stemmer et al [10] comparedlean chow-fedWistar rats with HFD-fed rats that were eithersensitive or partially resistant to diet-induced obesity afterelevenmonths ofHFD (40butter fat) A positive correlationwas established between levels of adiposity and the severityof renal damage Their results indicate that lipotoxicity isnot a strong contributor to renal dysfunction as they foundthat plasma triglyceride and free fatty acid as well as renaltriglyceride levels did not differ significantly between thetreatment and control groups [10]

While long-term feeding protocols are found in theliterature the effects of shorter-term feeding protocols tomimic early onset pathological changes within the kidneyfollowing high fat feeding are limited A variety of rodentmodels have been used to investigate the effects of consumingHFD over the course of eight weeks to 11 months [5 8 10 11]and although the effects of HFD on kidney morphology havebeen studied at four weeks [12] to our knowledge thereare currently no studies that have examined the effects ofshorter-term intake of high fat (as opposed to combinedhigh carbohydrate high fat) diets on renal function Forthis reason the present study was designed to examine theshort-term effects of feeding rats a HFD (60 fat) for sixweeks on renal morphology and biomarkers of functionPrior studies have shown that young (15-month-old) maleSprague-Dawley rats fed on a HFD (60 kcal from fat) foras few as six weeks develop indices of metabolic syndromeand cardiovascular disease increased visceral adiposityhyperglycemia impaired glucose tolerance hyperleptinemiasystemic inflammation (plasma TNF120572) lipid peroxidation(TBARS) and impaired endothelium-dependent vasodila-tion [13 14] Other studies have shown that increased visceraladiposity endothelial dysfunction hypertension inflamma-tion and oxidative stress (characteristics shared by theseanimals) increase blood pressure in the kidneys and promoterenal damage although this has yet to be examined in the 6-week HFD rat modelTherefore exploring the effect of short-term HFD on the kidneys will fill a gap in the literature andallow for a better understanding of early pathological changesthat occur due to consuming a HFD It was hypothesized thatsix weeks of high fat intake (60 fat) would lead to earlystages of renal disease in 15-month-oldmale Sprague-Dawleyrats as evidenced by morphological and functional changesin the kidney compared to control rats on a standard rodentchow diet (5 fat)

2 Materials and Methods

21 Animal Models Prior studies in our laboratory haveshown that feeding young male (15-month-old) Sprague-Dawley rats (140ndash160 g bodyweightHarlanTeklad Industries(MadisonWIUSA)) aHFD (60kcal from fat Cat numberD12492 Research Diets Inc New Brunswick NJ USA) forsix weeks results in symptoms associated with metabolicsyndrome including increased adiposity hyperleptinemiahyperglycemia endothelial dysfunction and hypertension incomparison to animals fed on standard rodent chow [13 14]The current study examined isolated kidneys plasma andurine samples from a subset of animals examined in theseprior IACUC-approved studies from September 2009 to May2010 Blood samples were obtained via cardiac puncture andcentrifuged at 13000 rpm at 4∘C for 15 minutes to separateformed elements from the plasma which was then storedat minus80∘C until analyses Urine was collected with a 25-gauge needle inserted directly into the bladder The urinewas then centrifuged at 13000 rpm at 4∘C for 10 minutesto remove debris and the supernatant was then storedat minus80∘C until analyses Following a midline laparotomykidneys were removed and stored at minus80∘C until analyseswhile a separate subset was embedded in Optimal CuttingTemperature (OCT Cat number 4583 Sakura Finetek USAInc Torrance CA USA) compound prior to freezing inisopentane cooled by liquid nitrogen and storing at minus80∘C

22 Morphometrics Animal morphometrics (body massepididymal fat padmass waist circumference and tail length)and renal tissue masses were measured Tail length is usedas marker of overall growth in rats [15] Epididymal fat padmass was used to determine variations in adiposity betweenHFDand chow-fed rats because the epididymal fat pad can beeasily removed from the animal objectively Moreover recentstudies have shown that this fat pad is highly correlated (1198772 =094) with total fat volume from the base of the skull to thedistal tibia as measured by in vivomicroCT scans [16]

A subset of frozen kidneys (119899 = 4 chow and 119899 = 5HFD rats) embedded in OCT compound were sectionedusing a cryostat (Leica Biosystems CM1950 Buffalo GroveIL USA) and sections (14 120583m) were collected onto (+)-glass microscope slides (Histobondcopy adhesive slides VWRVistaVision Radnor PA USA) Sections were allowed to air-dry for 10mins and were then fixed with 10 formalin foran additional 10mins Following 3-4 washes with tap watersectionswere stainedwithMayerrsquos hematoxylin (Cat number6194A16 Hardy Diagnostics Santa Maria CA USA) for2mins and then rinsed with warm tap water The slides werethen dipped in acid alcohol (05 HCl in 70 ethanol) andwashed in Scotts Tap Water Substitute (Cat number 26070-06 Electron Microscopy Sciences Hatfield PA USA) for5mins followed by a rinse with deionized water The sectionswere then counterstained with Eosin Y (Cat number IS4054Aldon Corporation Avon NY USA) for 3mins followed bya rinse with tap water and coverslips were mounted onto theglass microscope slides using SHURMount (Cat numberLC-W Triangle Biomedical Sciences Durham NC USA)Sections were viewed using a light microscope (Olympus

Journal of Nutrition and Metabolism 3

BX50) and images collected with an Olympus DP70 camera(Melville NY USA) to assess morphology

23 Biomarkers of Renal Function Measuring the excretionof total proteins in the urine proteinuria is a marker of earlyrenal disease [3 4] In fact multiple studies have found astronger association between proteinuria and renal diseaseoutcomes than any other tested factors [4] Moreover clinicalstudies on humans have identified proteinuria as amongthe first clinical symptoms of kidney damage [17 18] Totalurinary protein concentrations were measured on a randomset (119899 = 8group) using the Bradford technique (Cat number500-0006 Bio-Rad Hercules CA USA) Urinary creatinineconcentrations were measured on the same set of urinesamples (119899 = 8group) using an available kit (Cat numberCR01 Oxford Biomedical Research Rochester Hills MI) andvalues were used to calculate the protein creatinine ratioInterestingly creatinine clearance is also correlated with riskof renal failure [19]

Cystatin C produced by nucleated cells is filtered by theglomerulus and then catabolized by tubular cells in the kidney[20] For this reason plasma concentrations of cystatin Ccan be used as a biomarker of glomerular filtration rate [20]Urinary changes in cystatin C can be used asmarkers of acuterenal injury although not often used in CKD assessment [21]Serum creatinine concentrations are likewise often assessedas a marker of renal function however more recent studiesindicate that serum or plasma cystatin C is a stronger andmore consistent surrogate indicator of glomerular filtrationrate and renal function [19ndash27] Concentrations of cystatinC in the serum increase as much as 1-2 days prior toserum creatinine in conditions of acute kidney injuries [21]as well as in individuals with type 2 diabetes who havenormoalbuminuria [28] While serum creatinine levels canbe affected by sex age diet muscle mass and body masscystatin C levels are independent of gender muscle massand malignancy [21 27] Plasma cystatin C was thereforemeasured on a subset of samples (119899 = 10 per group) using anavailable ELISA kit (Cat number MSCTCO RampD SystemsMinneapolis MN USA) according to the manufacturerrsquosprotocol

Urinary H2O2 a biomarker of inflammation oxidative

stress and renal function was likewise measured on a subsetof samples (119899 = 6 per group) using an available kit (Catnumber ab102500 Abcam Cambridge MA USA) Crea-tinine concentrations were measured from the same urinesamples (Cat number CR01 Oxford Biomedical ResearchRochester Hills MI USA) and the urinary H

2O2 creatinine

ratio was calculated Urinary and tissue H2O2levels are often

examined in studies of renal function The increased renalperfusion pressure that is associated with hypertension hasbeen found to elevate excretion of H

2O2[29] SinceH

2O2can

stimulate proteinuria it is sometimes used as an indicator ofthe initiation of renal pathology [30]

24 Inflammatory Markers Chronic inflammation is oftenassociated with multiple disease states and has been hypoth-esized to contribute to both morbidity and mortality in CKDpatients [31] Studies on animals fed on HFD have found

lipid accumulation in glomeruli and proximal tubules withinthe kidneys along with increased expression of inflammatorymarkers particularly tumor necrosis factor alpha (TNF120572)and interleukin-6 (IL-6) [5 32] TNF120572 production is elevatedwith high fat intake and may in part cause insulin resistance[33] and both inflammatory cytokines can predict risk forchronic kidney disease [32] Briefly a subset of kidneys fromboth chow and HFD rats were transferred to a ground glasshomogenizer containing Tris-HCI buffer (10mM Tris (pH76 Cat number 161-0716 BioRad Hercules CA USA)1mM EDTA 1 triton X-100 01 sodium deoxycholate003 protease inhibitor cocktail (Cat number P2714 Sigma-Aldrich St Louis MO USA) and 1mM phenylmethane-sulfonyl fluoride (PMSF)) Samples were then centrifugedat 14000 rpm for 10 minutes at 4∘C to remove insolubledebris Total protein concentrations of the supernatants weredetermined using the Bradford Technique (Cat number 500-0006 Bio-Rad) Each sample (100 120583g total protein)wasmixedwith 6 120583L 5x sample buffer (06mL 1M Tris-HCl pH 685mL 50 glycerol 2mL 10 sodium dodecyl sulfate 1mL1 bromophenol blue 09mL deionized water and 2 120573-mercaptoethanol as a reducing agent) and then boiled for 3minutesThemixturewas then resolved using 4ndash15 gradientTris-HCl SDS-PAGE gels (Cat number 456-1083 Bio-Rad)and transferred to PVDFmembranes (Cat number 152-0176Bio-Rad) for 90 minutes at 200V Membranes were thenblocked for two hours in Tris buffered saline containing005 Tween 20 3 BSA fraction V and 5 nonfat milkfollowed by an overnight incubation at 4∘C in primaryantibodies for IL-6 (1 250 Cat number ab6672 AbcamCambridge MA USA) TNF120572 (1 500 Cat number 11948Cell Signaling Danvers MA USA) or the loading controlbeta-actin (1 2000 Cat number ab8227 Abcam CambridgeMAUSA) Primary antibodieswere prepared inTris bufferedsaline with 005 Tween 20 (TTBS) Membranes were thenexposed to anti-rabbit IgG secondary antibody (1 1000 forIL-6 and TNF120572 and 1 2000 for beta actin Cat number7074S Cell Signaling Technology Danvers MA) in TTBSfor one hour at room temperature followed by exposureto Pierce enhanced chemiluminescence western blottingsubstrate (Cat number 32279 Thermo Scientific RockfordIL USA) for 1min Immunoreactive bands were visualizedby exposure to X-ray film (Cat number 34090 ThermoScientific Rockford IL USA) and analyzed using NIHImageJ software Protein expression of IL-6 and TNF120572 werenormalized to beta-actin to determine the level of IL-6 (119899 = 5chow and 6 HFD animals) or TNF120572 (119899 = 8group) in eachkidney sample

25 Oxidative Stress Kidneys are especially susceptible tooxidative stress due to a high concentration of long-chainpolyunsaturated fatty acids which easily undergo lipid perox-idation when exposed to reactive oxygen species [34] Lipidperoxidation forms malondialdehyde as an end productwhich can be detected via a thiobarbituric acid reactive sub-stance (TBARS) assay a marker of oxidative damage that isoften examined A subset of samples (119899 = 5group) were usedto evaluate renal lipid peroxidation using a commercially


Table 1 Morphometric measurements

Measure Chow (119899) HFD (119899) 119901 valueBody mass (g) 3498 plusmn 798 (9) 3764 plusmn 103 (8) 0056Epididymal fat pad mass (g) 349 plusmn 018 (9) 560 plusmn 038 (8) lt0001Waist circumference (cm) 1659 plusmn 030 (9) 1796 plusmn 027 (8) 0004Tail length (cm) 201 plusmn 030 (9) 206 plusmn 032 (8) 0276Renal mass (g) 105 plusmn 004 (9) 119 plusmn 004 (8) 0019Data expressed as mean plusmn SEM and analyzed by Studentrsquos 119905-tests

200x 100x

Figure 1 Representative images of hematoxylin and eosin stained renal tissue sections from HFD animals Hematoxylin and eosin stainingshows no signs of morphological damage

available TBARS assay kit (Cat number 0801192 Zep-toMetrix Corporation Buffalo NY USA)

8-Hydroxy-21015840-deoxyguanosine (8-OHdG) is a sensitivebiomarker of oxidative stress in tissue and bodily fluids andis a major product of damage caused by oxidative stress toDNA 8-OHdG is produced by enzymatic cleavage of theguanine base the base most prone to oxidation Plasmasamples (119899 = 10 chow 119899 = 11 HFD) were filtered with a30 kDa ultrafilter (Cat number UFC503096 EMDMilliporeBillerica MA USA) and 8-hydroxyguanosine 8-OHdG and8-hydroxyguanine were examined as biomarkers of oxidativeDNA and RNA damage using a commercially available kit(Cat number 589320 Cayman Chemical Ann Arbor MIUSA) as oxidative stress has been shown to be elevatedduring early renal failure

26 Statistical Analyses Data are expressed as mean plusmn SEMStatistical analyses were computed using SigmaPlot (SystatSoftware Inc Version 130 San Jose CA USA) Data weretested for normality and then analyzed using Studentrsquos 119905-testsor the Mann-Whitney 119880 tests as appropriate 119901 values of le005 were considered significant

3 Results

31 Morphometrics Although the HFD rats tended to weighmore than the chow-fed rats this difference was not sta-tistically significant for the animals examined in this study(Table 1) Rats fed on a HFD demonstrated significantlyincreased epididymal fat pad mass compared to chow-fedanimals establishing that adipositywas increased by theHFD(Table 1) The waist circumferences of the HFD animals were

significantly increased when compared to chow-fed controlsindicating that the HFD increased abdominal adiposity(Table 1) Tail lengths a marker of overall growth were notsignificantly different (Table 1) The renal masses of the HFDrats were significantly greater compared to the renal massesof animals in the chow group (Table 1)

Morphological analyses using hematoxylin and eosin-stained tissue sections showed no structural differencesbetween the chow and HFD groups indicating that althoughthe mass of the HFD kidneys was increased damage to themicrostructure of the kidneys was not evident (Figure 1)

32 Biomarkers of Renal Function The urine protein creat-inine ratios of the chow and HFD rats did not differsignificantly indicating that the filtering ability of the HFDrat kidneys was not damaged (Table 2) There was alsono difference in the urine creatinine concentrations of thetwo groups (Table 2) Although HFD plasma cystatin Cconcentrations tended to be higher than chow rats thedifference was not sufficient to merit statistical signifi-cance (Table 2) Urinary hydrogen peroxide concentrationswere significantly increased in the HFD animals Howeverthe H

2O2 creatinine ratios were not significantly different

between the chow and HFD groups (Table 2)

33 Inflammatory Markers Western blot analyses of renaltissue established no significant difference in the TNF120572 pro-tein expression of the chow and HFD rat kidneys (Figure 2)Quantification of western blots of IL-6 expression in renaltissues likewise showed no difference between the kidneysfrom chow and HFD rats (Figure 3)


Table 2 Plasma and urinary markers of renal functionand damage

Measure Chow (119899) HFD (119899) 119901 valueUrine creatinine (mgL) 7650 plusmn 1106 (8) 9003 plusmn 1374 (8) 0456Urine protein creatinine ratio (AU) 601 plusmn 286 (8) 389 plusmn 131 (8) 0574Urine H2O2 (pM120583L) 0023 plusmn 0003 (6) 0048 plusmn 0005 (6) 0002Urine H2O2 creatinine ratio (AU) 0014 plusmn 0002 (6) 0026 plusmn 0007 (6) 0142Plasma cystatin C (120583gmL) 1435 plusmn 099 (10) 162 plusmn 112 (10) 0233Renal TBARS (mML) 269 plusmn 202 (5) 2808 plusmn 080 (5) 0600Plasma oxDNARNA (pgmL) 887 plusmn 145 (10) 1003 plusmn 188 (11) 0634Data expressed asmeanplusmn SEMand analyzed by Studentrsquos 119905-tests with the exception of urine protein creatinine ratio whichwas analyzed by theMann-Whitney119880 test

HFD Chow

Chow HFD000

002

004

006

008

010

012

014

016

(+)

120573-actin

TNF120572

TNF120572

dens

itom

etry

(AU

)Ki

dney

Figure 2 Renal tissue TNF120572 protein expression in chow and HFDratsThere was no difference in the TNF120572 expression of the two dietgroups (119899 = 8group) Retroperitoneal adipose tissue from a HFDratwas used as a positive control (+) and is shown in the first columnData were analyzed by Studentrsquos 119905-tests and are expressed as meansplusmn SEM 119901 = 0803

34 Oxidative Stress Markers Renal TBARS a measure oftissue oxidative stress via lipid peroxidation was not signif-icantly elevated in the HFD rats (Table 2) Plasma levels ofoxidized DNA and RNA quantified as plasma levels of mul-tiple oxidative stress markers including 8-hydroxyguanosinefrom RNA 8-OHdG from DNA and 8-hydroxyguaninewere examinedTherewere no significant differences betweenthe chow and HFD rat groups (Table 2)

4 Discussion

Young (15-month-old) male Sprague-Dawley rats fed onHFD (60 fat) for six weeks developed significant increasesin waist circumference and epididymal fat pad mass alongwith a trend towards increased bodymass (Table 1)Therewasno significant difference in the tail lengths of the animals onthe HFD and chow diets (Table 1) indicating that the animalswere of similar body sizeThese data also indicate that the rats

ChowHFD HFD

Chow HFD

Kidn

ey IL

-6 d

ensit

omet

ry (A

U)

000

005

010

015

020 (+)120573-actin

IL-6

Figure 3 Renal tissue IL-6 protein expression in chow and HFDrats There was no difference in the IL-6 expression of the two dietgroups (119899 = 5 chow and 6 HFD rats) Retroperitoneal adipose tissuefrom a HFD rat was used as a positive control (+) and is shownin the last column Data were analyzed by Studentrsquos 119905-tests and areexpressed as means plusmn SEM 119901 = 0903

used for the present study developed increased adiposityTheuse of an animal model that mimics the effects of metabolicsyndrome and prediabetes is consistent with prior studiesof HFD intake by both mice and rats [5 8 33 35ndash39]Previous studies from our laboratory also demonstrate that6 weeks of HFD results in hyperglycemia impaired glucosetolerance hypertension endothelial dysfunction oxidativestress (plasma TBARS) and inflammation (plasma TNF120572) inthese animals indicating that the HFD rats are alsomodels ofmetabolic syndrome [13 14]

The renal masses of the HFD rats examined in the presentstudy were significantly higher than the chow rats (Table 1)The lack of difference in tail lengths (Table 1) signifies thatthe increased mass was not caused by overall growth ofthe animals and instead suggests structural damage to thekidneys Altunkaynak et al [8] found similar increases inbody and renal masses of rats fed on HFD (30 fat) for12 weeks The increased renal mass may be attributed tohyperglycemia as proliferation and hypertrophy of mesangialcells and thickening of the glomerular basement membranepromote increased renal mass [40] along with vasodilation


inflammation and increased connective tissue [8] Despitethe increased overall mass of the kidneys hematoxylin andeosin staining of the HFD kidneys showed no evidenceof morphological damage (Figure 1) This is in contrast tolong-term studies of HFD that have been shown to causemorphological renal changes such as glomerular capillarydilation enlarged lumens in the tubules and Bowmanrsquos cap-sule amassing of extracellular proteins nephrondegradationglomerular membrane thickening glomerulosclerosis renaland tubular interstitial cell necrosis and shortened tubularepitheliums [5 8 41]

Proteinuria indicates that the filtering ability of theglomerulus is compromised and allowing proteins whichnormally remain in the blood to be passed through the fil-trationmembrane and excreted In evaluating renal functionalbumin is sometimes specifically examined however bystudying total protein excretion a wider picture of filtrationability can be ascertained Proteinuria specifically can beused as a marker of endothelial dysfunction as well as asurrogate outcome for renal disease progression Urinaryprotein concentrationswere normalized to urinary creatinineconcentrations in order to account for variations in urineoutput [42] Creatinine is also filtered by the kidneys andwas therefore evaluated for differences between the HFD andchow-fed groups Ruggiero et al [36] found no significantchanges in creatinine levels after C57BL mice consumed a45 fat diet for sixteen weeks the authors mentioned thisindicated early stages of renal damage without severe func-tional damage (Table 2) In the present study no statisticallysignificant differences were observed between the urinaryprotein to creatinine ratio for animals ingesting the HFD ascompared to the chow-fed animals (Table 2) indicating thatthe filtration abilities of the glomeruli were not damaged bytheHFD or at least not to the extent of allowing large proteinsto be excreted With a longer duration feeding protocolresearch suggests that the HFD may result in sustainedelevations in blood glucose concentrations further leading toincreased stress on the kidneys

Plasma cystatin C is considered a marker of early renaldysfunction and the lack of difference between the cystatin Cconcentrations of the HFD and chow-fed rats indicates thatthe six-week feeding protocolmay not have been long enoughto initiate functional renal damage (Table 2) The HFDgroup did develop higher concentrations of plasma cystatinC however this difference was not statistically significant(Table 2) Although cystatin C levels in plasma have beenshown to be an early sensitive marker of renal dysfunctionthese results indicate that the kidneys of the HFD-fed ratswere not damaged enough so as to prevent cystatin C frombeing filtered [22ndash24 43] Muntner et al [44] demonstratedthat serum cystatin C can be elevated in subjects who donot exhibit signs of micro- or macroalbuminuria or kidneydisease Rather the authors concluded that serum cystatinC was correlated with cardiovascular disease in overweightsubjects [44] Therefore plasma cystatin C alone may not bean accurate estimation of GFR

H2O2is an early marker of oxidative stress and inflam-

mation Despite the increased renal perfusion pressurethat is associated with hypertension and elevated H

2O2

excretion there was no significant difference in urinaryH2O2 creatinine ratio between the two groups (Table 2)

Elevated H2O2in the HFD group would have indicated early

renal damage as H2O2has been found to be elevated prior

to GFR decline proteinuria and more robust inflammatoryand fibrotic responses following high fat feedings [30 37 38]Overall these results demonstrate that although prior studiesindicate that the HFD rats develop plasma and vascularoxidative stress and inflammation in addition to endothelialdysfunction [13 14] these pathologies were not sufficient toimpair renal function

Western blot analyses established no significant dif-ference between the TNF120572 concentrations of renal tissueisolated from HFD and chow-fed rats (Figure 2) Althoughincreased TNF120572 expression in adipose tissue has been shownto promote to insulin resistance in overweight and obeserodent models [45 46] there was no indication that theHFD increased renal tissue expression of this inflammatorycytokine (Figure 2) There was also no increase in IL-6expression (Figure 3) a proinflammatory cytokine Otherexperimental models of renal injury indicate that increasesin IL-6 secretion are not necessarily an integral step inprogressive renal failure [47] Overall the lack of significantfindings in markers of early renal dysfunction such asproteinuria and urinary H

2O2 supports the lack of increases

in cytokine expression Chronic increased IL-6 levels are anindication of continued TNF120572 production and activation ofinflammatory pathways Despite the controversy regardingthe differing effects of IL-6 as a cytokine and myokine itcontinues to be studied as a marker of inflammation inHFD-induced obesity Studies on both overweight and obesewomen have found that serum TNF120572 and IL-6 levels werepositively correlated [48] while Ozay et al [35] found thatHFD-induced obesity in male Wistar albino rats (22 fat for12weeks) increased plasmaTNF120572without altering plasma IL-6 Conversely Kaya et al [49] found that IL-6 levels in obesehuman subjects were higher in both plasma and adiposetissue compared to TNF120572 Stemmer et al [10] examined theeffect of HFD (40 of calories from butter fat for 11 months)onmaleWistar rats and the creation of an inflammatory renalenvironment Their results indicate an increase of IL-6 andTNF120572 in retroperitoneal fat and in the kidneys while therewas no increase in circulating IL-6

Previous studies of the 6-week HFD rats showed thatplasma TBARS a measure of whole body oxidative stresswas increased in comparison to chow-fed controls [13]Despite the increased plasma TBARS renal TBARS werenot significantly different between chow and HFD-fed rats(Table 2) These results indicate that although the HFD ratsexhibited systemic oxidative stress oxidative stress within thekidneys is not yet evident Moreover no significant differencebetween oxidative DNARNA damage between the experi-mental and control groups was observed (Table 2) Oxidativestress examined via TBARS and DNARNA oxidation ispositively correlated [50] The lack of significant TNF120572 andIL-6 expression in renal tissue examined in the present studyis consistent with the observed lack of oxidative stress withinthe kidneys as inflammation and oxidative stress are typicallycorrelated [51]


5 Conclusions

In conclusion aside from morphological changes the onlymajor statistically significant findings in this study were anincrease in visceral adiposity and renal mass following ashort-term HFD This study is novel in that it is the firstto examine a HFD for only six weeks prior to this studythe shortest feeding protocol that is evident in the literatureis four weeks at which point only morphological changeswere examined as opposed to measures of renal functionPrevious research conducted on the six-week HFD rat modelshowed evidence of metabolic syndrome including signifi-cant vascular oxidative stress and inflammation endothelialdysfunction hypertension hyperglycemia hyperleptinemiaand impaired glucose tolerance [13 14]

It is possible that the methods used in this study mightnot have been sensitive enough to detect subtle changes inrenal function In prior studies by our laboratory 6 weeksof HFD significantly increased plasma concentrations of theketone beta-hydroxybutyrate from 472plusmn031 in chow rats to718 plusmn 077mgdL in the HFD animals [14] Considering thatketogenic diets can reduce renal responses to glucose it is alsopossible that the HFD exerts a protective effect in the kidney[51]This was shown in a prior study of mousemodels of type2 diabetes with nephropathy in which 8 weeks of consuminga ketogenic diet fully normalized albumin creatinine ratiosand the expression of stress-related genes [51] Therefore itis apparent from the current study that prolonged hyper-glycemia and inflammation are likely necessary to inducesignificant changes within the kidneys or that HFD mayexert some protective effects through increased generation ofketones

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was funded by a Graduate Research SupportProgram Award from ASU (Catherine Crinigan) as well as aSigmaXiGrant-in-Aid of Research (CatherineCrinigan)Theauthors would also like to thankKristin Ricklefs (ASU)MarcGirard (University of Poitiers France) and Zoha Ahmed(ASU) for technical assistance with the study

References

[1] Centers for Disease Control and Prevention National DiabetesStatistics Report Estimates of Diabetes and Its Burden in theUnited States 2014 US Department of Health and HumanServices Atlanta Ga USA 2014

[2] A T Reutens ldquoEpidemiology of diabetic kidney diseaserdquoMedical Clinics of North America vol 97 no 1 pp 1ndash18 2013

[3] W F Keane and G Eknoyan ldquoProteinuria albuminuria riskassessment detection elimination (PARADE) a position paperof the National Kidney Foundationrdquo The American Journal ofKidney Diseases vol 33 no 5 pp 1004ndash1010 1999

[4] A S Levey D Cattran A Friedman et al ldquoProteinuria asa surrogate outcome in CKD report of a scientific workshopsponsored by the National Kidney Foundation and the USFood and Drug Administrationrdquo American Journal of KidneyDiseases vol 54 no 2 pp 205ndash226 2009

[5] N Deji S Kume S-I Araki et al ldquoStructural and functionalchanges in the kidneys of high-fat diet-induced obese micerdquoAmerican Journal of Physiology Renal Physiology vol 296 no1 pp F118ndashF126 2009

[6] G M Reaven ldquoThe metabolic syndrome time to get off themerry-go-roundrdquo Journal of Internal Medicine vol 269 no 2pp 127ndash136 2011

[7] M C Foster S-J Hwang S A Porter J M Massaro U Hoff-mann and C S Fox ldquoFatty kidney hypertension and chronickidney disease the framingham heart studyrdquoHypertension vol58 no 5 pp 784ndash790 2011

[8] M E Altunkaynak E Ozbek B Z Altunkaynak I Can DUnal and B Unal ldquoThe effects of high-fat diet on the renalstructure and morphometric parametric of kidneys in ratsrdquoJournal of Anatomy vol 212 no 6 pp 845ndash852 2008

[9] T M Dwyer H L Mizelle K Cockrell and P Buhner ldquoRenalsinus lipomatosis and body composition in hypertensive obeserabbitsrdquo International Journal of Obesity and Related MetabolicDisorders vol 19 no 12 pp 869ndash874 1995

[10] K Stemmer D Perez-Tilve G Ananthakrishnan et al ldquoHigh-fat-diet-induced obesity causes an inflammatory and tumor-promotingmicroenvironment in the rat kidneyrdquoDiseaseModelsamp Mechanisms vol 5 no 5 pp 627ndash635 2012

[11] R Buettner K G Parhofer M Woenckhaus et al ldquoDefininghigh-fat-diet rat models metabolic and molecular effects ofdifferent fat typesrdquo Journal of Molecular Endocrinology vol 36no 3 pp 485ndash501 2006

[12] A Pranprawit F M Wolber J A Heyes A L Molan andM C Kruger ldquoShort-term and long-term effects of excessiveconsumption of saturated fats andor sucrose on metabolicvariables in Sprague Dawley rats a pilot studyrdquo Journal of theScience of Food and Agriculture vol 93 no 13 pp 3191ndash31972013

[13] K L Sweazea M Lekic and B R Walker ldquoCompari-son of mechanisms involved in impaired vascular reactivitybetween high sucrose and high fat diets in ratsrdquo Nutrition andMetabolism vol 7 article 48 2010

[14] K L Sweazea and B R Walker ldquoHigh fat feeding impairsendothelin-1 mediated vasoconstriction through increasediNOS-derived nitric oxiderdquo Hormone and Metabolic Researchvol 43 no 7 pp 470ndash476 2011

[15] L Gonzalez Bosc M L Kurnjek A Muller and N BassoldquoEffect of chronic angiotensin II inhibition on the cardio-vascular system of the normal ratrdquo The American Journal ofHypertension vol 13 no 12 pp 1301ndash1307 2000

[16] Y K Luu S Lublinsky E Ozcivici et al ldquoIn vivo quantificationof subcutaneous and visceral adiposity by micro-computedtomography in a small animal modelrdquo Medical Engineering ampPhysics vol 31 no 1 pp 34ndash41 2009

[17] N Kambham G S Markowitz A M Valeri J Lin andV D DrsquoAgati ldquoObesity-related glomerulopathy an emergingepidemicrdquo Kidney International vol 59 no 4 pp 1498ndash15092001

[18] R D Adelman I G Restaino U S Alon and D L BloweyldquoProteinuria and focal segmental glomerulosclerosis in severelyobese adolescentsrdquoThe Journal of Pediatrics vol 138 no 4 pp481ndash485 2001


[19] M Praga E Hernandez E Morales et al ldquoClinical featuresand long-term outcome of obesity-associated focal segmentalglomerulosclerosisrdquo Nephrology Dialysis Transplantation vol16 no 9 pp 1790ndash1798 2001

[20] AGrubb ldquoDiagnostic value of analysis of cystatinC andproteinHC in biological fluidsrdquoClinicalNephrology vol 38 supplement1 pp S20ndashS27 1992

[21] J C Sirota J Klawitter andC L Edelstein ldquoBiomarkers of acutekidney injuryrdquo Journal of Toxicology vol 2011 Article ID 32812010 pages 2011

[22] J S C Chew M Saleem C M Florkowski and P MGeorge ldquoCystatin C a paradigm of evidence based laboratorymedicinerdquo The Clinical Biochemist Reviews vol 29 no 2 pp47ndash62 2008

[23] J S C Chew-Harris C M Florkowski P M George J LElmslie and Z H Endre ldquoThe relative effects of fat versusmuscle mass on cystatin C and estimates of renal function inhealthy youngmenrdquoAnnals of Clinical Biochemistry vol 50 no1 pp 39ndash46 2013

[24] R Hojs S Bevc R Ekart M Gorenjak and L PuklavecldquoSerum cystatin C as an endogenous marker of renal functionin patients with chronic kidney diseaserdquo Renal Failure vol 30no 2 pp 181ndash186 2008

[25] N V McNamara R Chen M R Janu P Bwititi G Car andM Seibel ldquoEarly renal failure detection by cystatin C in Type 2diabetes mellitus varying patterns of renal analyte expressionrdquoPathology vol 41 no 3 pp 269ndash275 2009

[26] V RDharnidharka CKwon andG Stevens ldquoSerumcystatinCis superior to serum creatinine as a marker of kidney functiona meta-analysisrdquo American Journal of Kidney Diseases vol 40no 2 pp 221ndash226 2002

[27] O Schuck V Teplan M Stollova and J Skibova ldquoEstimationof glomerular filtration rate in obese patients with chronicrenal impairment based on serum cystatin C levelsrdquo ClinicalNephrology vol 62 no 2 pp 92ndash96 2004

[28] Y K Jeon M R Kim J E Huh et al ldquoCystatin C as an earlybiomarker of nephropathy in patients with type 2 diabetesrdquoJournal of Korean Medical Science vol 26 no 2 pp 258ndash2632011

[29] C Jin C Hu A Polichnowski et al ldquoEffects of renal perfusionpressure on renalmedullary hydrogen peroxide and nitric oxideproductionrdquo Hypertension vol 53 no 6 pp 1048ndash1053 2009

[30] T Yoshioka I Ichikawa and A Fogo ldquoReactive oxygenmetabolites cause massive reversible proteinuria and glomeru-lar sieving defect without apparent ultrastructural abnormalityrdquoJournal of the American Society of Nephrology vol 2 no 4 pp902ndash912 1991

[31] H F Tbahriti D Meknassi R Moussaoui et al ldquoInflammatorystatus in chronic renal failure the role of homocysteinemia andpro-inflammatory cytokinesrdquoWorld Journal of Nephrology vol2 pp 31ndash37 2013

[32] C Ruster and G Wolf ldquoAdipokines promote chronic kidneydiseaserdquo Nephrology Dialysis Transplantation vol 28 no 4 pp8ndash14 2013

[33] S E Borst and C F Conover ldquoHigh-fat diet induces increasedtissue expression of TNF-120572rdquo Life Sciences vol 77 no 17 pp2156ndash2165 2005

[34] EOzbek ldquoInduction of oxidative stress in kidneyrdquo InternationalJournal ofNephrology vol 2012 Article ID465897 9 pages 2012

[35] R Ozay E Uzar A Aktas et al ldquoThe role of oxidative stressand inflammatory response in high-fat diet induced peripheral

neuropathyrdquo Journal of Chemical Neuroanatomy vol 55 pp 51ndash57 2014

[36] C Ruggiero M Ehrenshaft E Cleland and K Stadler ldquoHigh-fat diet induces an initial adaptation of mitochondrial bioener-getics in the kidney despite evident oxidative stress and mito-chondrial ROS productionrdquo American Journal of PhysiologyEndocrinology andMetabolism vol 300 no 6 pp E1047ndashE10582011

[37] A-E Decleves A V Mathew R Cunard and K SharmaldquoAMPK mediates the initiation of kidney disease induced by ahigh-fat dietrdquo Journal of the American Society of Nephrology vol22 no 10 pp 1846ndash1855 2011

[38] A E Decleves J J Rychak D J Smith and K SharmaldquoEffects of high-fat diet and losartan on renal cortical blood flowusing contrast ultrasound imagingrdquo The American Journal ofPhysiologymdashRenal Physiology vol 305 no 9 pp F1343ndashF13512013

[39] A H Stark B Timar and Z Madar ldquoAdaptation of SpragueDawley rats to long-term feeding of high fat or high fructosedietsrdquo European Journal of Nutrition vol 39 no 5 pp 229ndash2342000

[40] E N Obineche E Mensah-Brown S I Chandranath IAhmed O Naseer and A Adem ldquoMorphological changesin the rat kidney following long-term diabetesrdquo Archives ofPhysiology and Biochemistry vol 109 no 3 pp 241ndash245 2001

[41] T Thethi M Kamiyama and H Kobori ldquoThe link betweenthe renin-angiotensin-aldosterone system and renal injury inobesity and the metabolic syndromerdquo Current HypertensionReports vol 14 no 2 pp 160ndash169 2012

[42] S SWaikar V S Sabbisetti and J V Bonventre ldquoNormalizationof urinary biomarkers to creatinine during changes in glomeru-lar filtration raterdquo Kidney International vol 78 no 5 pp 486ndash494 2010

[43] G S Hotamisligil N S Shargill and B M SpiegelmanldquoAdipose expression of tumor necrosis factor-120572 direct role inobesity-linked insulin resistancerdquo Science vol 259 no 5091 pp87ndash91 1993

[44] P Muntner D Mann J Winston S Bansilal and M EFarkouh ldquoSerum cystatin C and increased coronary heartdisease prevalence inUS adults without chronic kidney diseaserdquoThe American Journal of Cardiology vol 102 no 1 pp 54ndash572008

[45] W P Cawthorn and J K Sethi ldquoTNF-120572 and adipocyte biologyrdquoFEBS Letters vol 582 no 1 pp 117ndash131 2008

[46] S A Jones D J Fraser C A Fielding and G W JonesldquoInterleukin-6 in renal disease and therapyrdquoNephrologyDialysisTransplantation vol 30 no 4 pp 564ndash574 2015

[47] S Fenkci S Rota N Sabir Y Sermez A Guclu and B AkdagldquoRelationship of serum interleukin-6 and tumor necrosis factoralpha levels with abdominal fat distribution evaluated by ultra-sonography in overweight or obese postmenopausal womenrdquoJournal of Investigative Medicine vol 54 no 8 pp 455ndash4602006

[48] PAKern S RanganathanC Li LWood andGRanganathanldquoAdipose tissue tumor necrosis factor and interleukin-6 expres-sion in human obesity and insulin resistancerdquoAmerican Journalof PhysiologymdashEndocrinology and Metabolism vol 280 no 5pp E745ndashE751 2001

[49] Y Kaya A Ceb N Soylemez H Dem H H Alp and E BakanldquoCorrelations between oxidative DNA damage oxidative stressand coenzyme Q10 in patients with coronary artery diseaserdquo


International Journal of Medical Sciences vol 9 no 8 pp 621ndash626 2012

[50] H K Vincent and A G Taylor ldquoBiomarkers and potentialmechanisms of obesity-induced oxidant stress in humansrdquoInternational Journal ofObesity vol 30 no 3 pp 400ndash418 2006

[51] M M Poplawski J W Mastaitis F Isoda F Grosjean FZheng and C VMobbs ldquoReversal of diabetic nephropathy by aketogenic dietrdquo PLoS ONE vol 6 no 4 Article ID e18604 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Deji et al [5] showed that feeding 6-week-oldmice a highfat diet (60 fat) for 12 weeks increased body mass plasmaglucose insulin and triglyceride concentrations and inducedhypertension and kidney disease as evidenced by increasedalbuminuria alterations in renal morphology and renal lipidaccumulation Similarly Altunkaynak et al [8] demonstratedthat feeding adult female Sprague-Dawley rats a moderate fatdiet (30 fat) for 12 weeks caused the animals to becomeoverweight and to develop increased kidney masses andvolumes with significant morphological changes indicativeof renal disease Moreover renal fat accumulation is evidentwith even moderate fat intake in rabbits (10 corn oil +5 lard for 8ndash12 weeks) [9] Stemmer et al [10] comparedlean chow-fedWistar rats with HFD-fed rats that were eithersensitive or partially resistant to diet-induced obesity afterelevenmonths ofHFD (40butter fat) A positive correlationwas established between levels of adiposity and the severityof renal damage Their results indicate that lipotoxicity isnot a strong contributor to renal dysfunction as they foundthat plasma triglyceride and free fatty acid as well as renaltriglyceride levels did not differ significantly between thetreatment and control groups [10]

While long-term feeding protocols are found in theliterature the effects of shorter-term feeding protocols tomimic early onset pathological changes within the kidneyfollowing high fat feeding are limited A variety of rodentmodels have been used to investigate the effects of consumingHFD over the course of eight weeks to 11 months [5 8 10 11]and although the effects of HFD on kidney morphology havebeen studied at four weeks [12] to our knowledge thereare currently no studies that have examined the effects ofshorter-term intake of high fat (as opposed to combinedhigh carbohydrate high fat) diets on renal function Forthis reason the present study was designed to examine theshort-term effects of feeding rats a HFD (60 fat) for sixweeks on renal morphology and biomarkers of functionPrior studies have shown that young (15-month-old) maleSprague-Dawley rats fed on a HFD (60 kcal from fat) foras few as six weeks develop indices of metabolic syndromeand cardiovascular disease increased visceral adiposityhyperglycemia impaired glucose tolerance hyperleptinemiasystemic inflammation (plasma TNF120572) lipid peroxidation(TBARS) and impaired endothelium-dependent vasodila-tion [13 14] Other studies have shown that increased visceraladiposity endothelial dysfunction hypertension inflamma-tion and oxidative stress (characteristics shared by theseanimals) increase blood pressure in the kidneys and promoterenal damage although this has yet to be examined in the 6-week HFD rat modelTherefore exploring the effect of short-term HFD on the kidneys will fill a gap in the literature andallow for a better understanding of early pathological changesthat occur due to consuming a HFD It was hypothesized thatsix weeks of high fat intake (60 fat) would lead to earlystages of renal disease in 15-month-oldmale Sprague-Dawleyrats as evidenced by morphological and functional changesin the kidney compared to control rats on a standard rodentchow diet (5 fat)

2 Materials and Methods

21 Animal Models Prior studies in our laboratory haveshown that feeding young male (15-month-old) Sprague-Dawley rats (140ndash160 g bodyweightHarlanTeklad Industries(MadisonWIUSA)) aHFD (60kcal from fat Cat numberD12492 Research Diets Inc New Brunswick NJ USA) forsix weeks results in symptoms associated with metabolicsyndrome including increased adiposity hyperleptinemiahyperglycemia endothelial dysfunction and hypertension incomparison to animals fed on standard rodent chow [13 14]The current study examined isolated kidneys plasma andurine samples from a subset of animals examined in theseprior IACUC-approved studies from September 2009 to May2010 Blood samples were obtained via cardiac puncture andcentrifuged at 13000 rpm at 4∘C for 15 minutes to separateformed elements from the plasma which was then storedat minus80∘C until analyses Urine was collected with a 25-gauge needle inserted directly into the bladder The urinewas then centrifuged at 13000 rpm at 4∘C for 10 minutesto remove debris and the supernatant was then storedat minus80∘C until analyses Following a midline laparotomykidneys were removed and stored at minus80∘C until analyseswhile a separate subset was embedded in Optimal CuttingTemperature (OCT Cat number 4583 Sakura Finetek USAInc Torrance CA USA) compound prior to freezing inisopentane cooled by liquid nitrogen and storing at minus80∘C

22 Morphometrics Animal morphometrics (body massepididymal fat padmass waist circumference and tail length)and renal tissue masses were measured Tail length is usedas marker of overall growth in rats [15] Epididymal fat padmass was used to determine variations in adiposity betweenHFDand chow-fed rats because the epididymal fat pad can beeasily removed from the animal objectively Moreover recentstudies have shown that this fat pad is highly correlated (1198772 =094) with total fat volume from the base of the skull to thedistal tibia as measured by in vivomicroCT scans [16]

A subset of frozen kidneys (119899 = 4 chow and 119899 = 5HFD rats) embedded in OCT compound were sectionedusing a cryostat (Leica Biosystems CM1950 Buffalo GroveIL USA) and sections (14 120583m) were collected onto (+)-glass microscope slides (Histobondcopy adhesive slides VWRVistaVision Radnor PA USA) Sections were allowed to air-dry for 10mins and were then fixed with 10 formalin foran additional 10mins Following 3-4 washes with tap watersectionswere stainedwithMayerrsquos hematoxylin (Cat number6194A16 Hardy Diagnostics Santa Maria CA USA) for2mins and then rinsed with warm tap water The slides werethen dipped in acid alcohol (05 HCl in 70 ethanol) andwashed in Scotts Tap Water Substitute (Cat number 26070-06 Electron Microscopy Sciences Hatfield PA USA) for5mins followed by a rinse with deionized water The sectionswere then counterstained with Eosin Y (Cat number IS4054Aldon Corporation Avon NY USA) for 3mins followed bya rinse with tap water and coverslips were mounted onto theglass microscope slides using SHURMount (Cat numberLC-W Triangle Biomedical Sciences Durham NC USA)Sections were viewed using a light microscope (Olympus







2O2 creatinine



2O2[29] SinceH

2O2can








200x 100x





3 Results











HFD Chow

Chow HFD000

002

004

006

008

010

012

014

016

(+)

120573-actin

TNF120572

TNF120572

dens

itom

etry

(AU

)Ki

dney



4 Discussion


ChowHFD HFD

Chow HFD

Kidn

ey IL

-6 d

ensit

omet

ry (A

U)

000

005

010

015

020 (+)120573-actin

IL-6










2O2










5 Conclusions





Acknowledgments


References





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















2O2 creatinine



2O2[29] SinceH

2O2can








200x 100x





3 Results











HFD Chow

Chow HFD000

002

004

006

008

010

012

014

016

(+)

120573-actin

TNF120572

TNF120572

dens

itom

etry

(AU

)Ki

dney



4 Discussion


ChowHFD HFD

Chow HFD

Kidn

ey IL

-6 d

ensit

omet

ry (A

U)

000

005

010

015

020 (+)120573-actin

IL-6










2O2










5 Conclusions





Acknowledgments


References





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















200x 100x





3 Results











HFD Chow

Chow HFD000

002

004

006

008

010

012

014

016

(+)

120573-actin

TNF120572

TNF120572

dens

itom

etry

(AU

)Ki

dney



4 Discussion


ChowHFD HFD

Chow HFD

Kidn

ey IL

-6 d

ensit

omet

ry (A

U)

000

005

010

015

020 (+)120573-actin

IL-6










2O2










5 Conclusions





Acknowledgments


References





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















HFD Chow

Chow HFD000

002

004

006

008

010

012

014

016

(+)

120573-actin

TNF120572

TNF120572

dens

itom

etry

(AU

)Ki

dney



4 Discussion


ChowHFD HFD

Chow HFD

Kidn

ey IL

-6 d

ensit

omet

ry (A

U)

000

005

010

015

020 (+)120573-actin

IL-6










2O2










5 Conclusions





Acknowledgments


References





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















2O2










5 Conclusions





Acknowledgments


References





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















5 Conclusions





Acknowledgments


References





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article short-term high fat intake does not...

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