research article preventive effect of salicylate and...

Research ArticlePreventive Effect of Salicylate and Pyridoxamine onDiabetic Nephropathy

Tarek Kamal Abouzed12 Seiichi Munesue1 Ai Harashima1 Yusuke Masuo3 Yukio Kato3

Khaled Khailo2 Hiroshi Yamamoto1 and Yasuhiko Yamamoto1

1Department of Biochemistry and Molecular Vascular Biology Kanazawa University Graduate School of Medical Sciences13-1 Takara-machi Kanazawa 920-8640 Japan2Department of Biochemistry Faculty of Veterinary Medicine Kafrelsheikh University Kafr El Sheikh 33516 Egypt3Molecular Pharmacotherapeutics Faculty of Pharmacy Kanazawa University Kakuma-machi Kanazawa 920-1192 Japan

Correspondence should be addressed to Yasuhiko Yamamoto yasuyamamedkanazawa-uacjp

Received 30 August 2016 Revised 31 October 2016 Accepted 7 November 2016

Academic Editor Carol A Witczak

Copyright copy 2016 Tarek Kamal Abouzed 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

Objective Diabetic nephropathy is a life-threatening complication in patients with long-standing diabetes Hemodynamicinflammatory and metabolic factors are considered as developmental factors for diabetic nephropathy In this study we evaluatedwhether pharmacological interventions with salicylate compared to pyridoxamine could prevent diabetic nephropathy in miceMethods Male mice overexpressing inducible nitric oxide synthase in pancreatic 120573-cells were employed as a diabetic modelSalicylate (3 gkg diet) or pyridoxamine (1 gL drinking water sim200mgkgday) was given for 16 weeks to assess the developmentof diabetic nephropathy Treatment with long-acting insulin (Levemir 2 unitskg twice a day) was used as a control ResultsAlthough higher blood glucose levels were not significantly affected by pyridoxamine early to late stage indices of nephropathywereattenuated including kidney enlargement albuminuria and increased serum creatinine glomerulosclerosis and inflammatoryand profibrotic gene expressions Salicylate showed beneficial effects on diabetic nephropathy similar to those of pyridoxaminewhich include lowering blood glucose levels and inhibiting macrophage infiltration into the kidneys Attenuation of macrophageinfiltration into the kidneys and upregulation of antiglycating enzyme glyoxalase 1 gene expression were found only in the salicylatetreatment group Conclusions Treatment with salicylate and pyridoxamine could prevent the development of diabetic nephropathyin mice and therefore would be a potentially useful therapeutic strategy against kidney problems in patients with diabetes

1 Introduction

Diabetic nephropathy is a life-threatening complication thatoccurs in 20ndash40 of patients suffering from diabetes andis the leading cause of chronic kidney disease [1] Thereare many factors influencing the development of diabeticnephropathy including genetic environmental hemody-namic and metabolic factors [2] Development of diabeticnephropathy is characterized by glomerular hyperfiltrationincreased excretion of urinary albumin and thickening ofglomerular basement membranes followed by an expansionof the extracellularmatrix in themesangial areas It ultimatelyprogresses to glomerular sclerosis associated with renaldysfunction [3]

Hyperglycemia per se is a major cause of the onset andprogression of diabetic complications High glucose con-centrations lead to vascular derangements through variousmolecular mechanisms including glycation polyol pathwayhexosamine pathway oxidative stress and activation of pro-tein kinase C [4] These factors interact with one anotherthereby facilitating host responses including inflammatoryreactions and stress responses leading to glomerulosclerosisunder diabetic conditions Of them our group and othersfocus on the glycation reaction and advanced glycation end-products (AGE) implicated in the pathogenesis of diabeticnephropathy [5 6]The receptor forAGE (RAGE) associationwith ligands triggers the acceleration of this disease [7]For example RAGE-overexpressing diabetic mice showed

Hindawi Publishing CorporationJournal of Diabetes ResearchVolume 2016 Article ID 1786789 10 pageshttpdxdoiorg10115520161786789

2 Journal of Diabetes Research

progressive glomerulosclerosis with renal dysfunction com-pared to diabetic littermates lacking the RAGE transgene [8]Moreover homozygous RAGE-null diabetic mice failed todevelop mesangial matrix expansion or glomerulosclerosis[9] Intracellular RAGE signaling involves activation of tran-scriptional factor NF120581B activity thus enhancing proinflam-matory reactions [10 11]

To prevent and treat diabetic kidney disease we examinedthe effects of pyridoxamine an antiglycating drug andsalicylate an anti-inflammatory drug on the developmentof diabetic nephropathy using our unique animal modelPyridoxamine is one of the natural forms of vitamin B6 and isknown to act as a nucleophilic trap for reactive carbonyl inter-mediates in AGE formation [12] Pyridoxamine is reportedto prevent the development of early stage nephropathy in arat model of diabetes [13 14] The effectiveness on late stagediabetic nephropathy including glomerulosclerosis is not yetfully elucidated in a rat model of diabetes [13] Salicylate is awell-known anti-inflammatory compound that inhibits I120581Bkinase-120573 (IKK120573) and is downstream of NF120581B [15] Recentclinical studies have shown that salsalate a nonacetylatedsalicylate lowered fasting blood glucose levels and HbA1c aswell as markers of inflammation [16 17] but did not decreasethe formation late glycation products [18]

In this study we studied the effects of salicylate comparedto pyridoxamine on early to late stages of diabetic nephropa-thy in our original mouse model of diabetic kidney injurieswith insulin treatment group as a control

2 Materials and Methods

21 Animals The male mice of a type 1 diabetic mouseline that overexpresses inducible NO synthase (iNOS) inpancreatic 120573-cells and control CD1 (ICR) CD-1 background(Charles River Japan) were used in this study [8 19]They were fed a high-calorie diet (Labo H standard NosanYokohma Japan) that contained 311 protein 82 lipids33 fiber 63 ash and 81 water The transgenic miceconsistently develop overt diabetes as early as 1 week afterbirth due to the NO-mediated selective destruction of insulinproducing pancreatic 120573-cells which results in advanceddiabetic nephropathy [8 19 20] Animals were treated inaccordance with the Fundamental Guidelines for ProperConducts of Animal Experiment and Related Activities inAcademic Research Institutions under the jurisdiction ofthe Ministry of Education Culture Sports Science andTechnology of Japan Animal experiments were approvedby the Committee on Animal Experimentation of KanazawaUniversity

22 Animal Groups with Different Treatment Pyridoxamine-dihydrochloride-monohydrate (4-aminomethyl-3-hydroxy-2-methyl-5-oxymethylpyridinedihydrochloride) was pur-chased from Tokyo Chemical Industry Co Japan Thepyridoxamine dihydrochloride was given at 1 gL (sim200mgkgday) in the drinking water from 4 to 20 weeks of age for16 weeks resulting in the serum concentration at 042 120583M(lt003 120583M in nontreated mice) Salicylate (sodium salicylate

Sigma-Aldrich Japan) was given at 3 gkg in their dietsyielding the serum concentration at 1026 120583M (not detectedin nontreated mice) For the insulin treatment group along-acting insulin analog of insulin detemir (Levemir NovoNordisk) was subcutaneously injected twice a day at the doseof 2 unitskg body weight

The following five animal groups were subjected tophenotypic analyses (1) nondiabetic control (119899 = 8) (2)diabetic mice without treatment (119899 = 19) (3) diabetic micewith insulin treatment (119899 = 8) (4) diabetic mice withsalicylate treatment (119899 = 12) and (5) diabetic mice withpyridoxamine treatment (119899 = 15)

23 Biochemical Assays Urinary samples were collectedby manual manipulation then kept in Eppendorf tubes atminus80∘C Blood glucose levels were determined using Glu-cocard (Arkray Japan) Body weight (BW) and nonfastingblood glucose levels were checked at 4 8 12 16 and 20weeks of age Serum and urinary creatinine levels weremonitored at 12 16 and 20 weeks of age The urinaryalbumin-creatinine ratio (ACR) was calculated at 20 weeksof age Serum and urinary creatinine were measured usingthe creatinine-test (Wako Osaka Japan) Urinary albuminand beta-2 microglobulin levels were measured by a mouseAlbumin ELISA Quantitation kit (AlbuWell II M ExocellPhiladelphia PA) and a mouse BMG1205732-MG ELISA kit(Elabscience Japan) respectively

24 Histopathology After sacrificing the animals at 20 weeksof age the left kidney was obtained and fixed in a Carnoyrsquossolution (10 acetic acid 60methanol and 3 chloroform)or 4 paraformaldehyde For 48 hr fixation paraformalde-hyde and Carnoyrsquos solutions were replaced by phosphatebuffer saline (PBS) and 70 methanol respectively Thekidney specimens were sectioned at 1 120583m thicknesses fol-lowed by the periodic acid-Schiff (PAS) periodic acid-methenamine silver (PAM) or hematoxylin and eosin (HampE)stain and imaged using a lightmicroscope (ZEISSmicroscopewith Fujifilm digital camera HC 300Z) Quantitative exami-nations were done on at least 50 glomeruli per mouse Theseverity of the renal sclerosis was scored by multiple analystson an arbitrary scale from 0 to 4 [8 9] The mean glomerulararea was determined as described previously [8 9]

25 Flow Cytometry (FACS) To evaluate macrophage infil-tration cells from the entire right kidney from each animalgroup at 20 weeks of age were subjected to FACS analysisThe kidney was cut into small pieces by scissors and digestedby incubating with collagenase II (1 120583gmL) Cells werethen washed and resuspended in staining buffer (PBS with2 FCS) containing FcBlock (BD Biosciences) Cells werestained by incubating for 15min at 4∘C in the dark with thefollowing antibodies CD45-PE (eBioscience) CD11bndashAPC-Cy7 (BD Biosciences) and F480-PE-Cy7 (BD Biosciences)Cells were resuspended in 200120583L of staining buffer con-taining 02120583gmLpropidium iodide (Sigma-Aldrich) filteredthrough a 100 120583m mesh and analyzed by FACSAria II (BDBiosciences) To quantify viable cell numbers liquid counting

Journal of Diabetes Research 3

Table 1 DNA primer pairs used in this study

Forward primer Reverse primerIl-1120573 51015840-GCT CAG GGT CAC AAG AAA CC -31015840 51015840- CAT CAA AGC AAT GTG CTG GT-31015840

Il-10 51015840- TGT TTC CAT TGG GGA CAC TT-31015840 51015840- AAC TGG CCA CAG TTT TCA GG-31015840

Tbp 51015840- ACC CTT CAC CAA TGA CTC CTA TG-31015840 51015840-TGA CTG CAG CAA ATC GCT TGG-31015840

Tol4 51015840- GGG TCA AGG AAC AGA AGC AG-31015840 51015840- GCT CAT TTC TCA CCC AGT CC-31015840

Mcp-1 51015840- TCC CAA TGA GTA GGC TGG AG-31015840 51015840- AAG TGC TTG AGG TGG TTG TG-31015840

Col1a1 51015840- GCT CCT CTT AGG GGC CAC T-31015840 51015840- ATT GGG GAC CCT TAG GCC AT-31015840

Glo1 51015840- TTG GTC ACA TTG GGA TTG CC-31015840 51015840- TCC TTT CAT TTT CCC GTC ATC AG-31015840

Mcp-2 51015840- GAA GAT GGT TAT CGT CAC CAC C-31015840 51015840- CGT TCC AGG CAT TGT ACC ACT-31015840

Ccr2 51015840- ATC CAC GGC ATA CTA TCA ACA TC-31015840 51015840- TCG TAG TCA TAC GGT GTG GTG-31015840

Vcam1 51015840- TTG GGA GCC TCA ACG GTA CT-31015840 51015840- GCA ATC GTT TTG TAT TCA GGG GA -31015840

beads were employed from the BD Cell Viability kit (BDBiosciences) Datawere transferred and analyzedwith FlowJosoftware (Tree Star)

26 RNA Extraction and Real-Time Polymerase Chain Reac-tion (PCR) Expression of mRNAwasmeasured by quantita-tive real-time polymerase chain reaction (qRT-PCR) Kidneytissues were snap-frozen in liquid nitrogen and preserved atminus80∘C prior to RNA extraction Total RNA was extractedusing Trizol reagent and purified by RNeasy plus kit (QIA-GEN) according to the manufacturerrsquos instructions Comple-mentary DNA was synthesized by using Oligo (dT) primerAffinityScript QPCR cDNA Synthesis Kit (Agilent Technolo-gies USA) according to the manufacturerrsquos instructions TheSYBR green qRT-PCR was performed with specific DNAprimers (Table 1) Amplification and real-time fluorescencedetectionwere performedusing amodelMx3005PReal-TimeQPCR system (Stratagene Products Division Agilent Tech-nologies) and the following protocol an initial denaturationand polymerase activation step for 2min at 95∘C followedby 40 cycles at 95∘C for 5 s and 60∘C for 20 s The mRNAexpression levels were normalized to TATA-binding protein(Tbp)

27 Determination of Salicylate and Pyridoxamine Concentra-tions inMouse Sera Serum samples (40 120583L) weremixed with5 120583L of 2120583M cimetidine (internal standard) 5120583L of waterand 150120583L of 200120583Macetonitrile followed by centrifugationat 22000timesg for 10min A 3 120583L aliquot of supernatant wasinjected into LC-MSMS system consisting of a NexeraHPLC system (Shimadzu Kyoto Japan) coupled to a triplequadrupole mass spectrometer (LCMS-8040 Shimadzu)Chromatography for salicylate was performed by means ofgradient elution (flow rate 04mLmin) as follows 0ndash10minute 99 A1 B 10ndash40 minutes 99 A1 B to 5A95 B 40ndash50 minutes 5 A95 B 50ndash51 minutes 5A95 B to 99 A1 B 51ndash70 minutes 99 A1 B [Awater containing 01 formic acid B methanol containing01 formic acid] using ACQUITY UPLC BEH C

18(21 times

100mm 17 120583m Waters Milford MA) at 50∘C Chromatog-raphy for pyridoxamine was performed by means of gradientelution (flow rate 04mLmin) as follows 0ndash10 minute 5

A95 B 10ndash60 minutes 5 A95 B to 50 A50 B60ndash65 minutes 50 A50 B to 65 A35 B 65ndash75minutes 65 A35 B 75ndash77 minutes 65 A35 B to 5A95 B 77ndash145 minutes 5 A95 B [A water containing01 formic acid B acetonitrile containing 01 formic acid]using SeQuant ZIC-cHILIC column (21 times 150mm 3 120583mMerckMillipore Darmstadt Germany) at 50∘CThemultiplereactionmonitoringwas set at 1691 to 1341 for pyridoxamine1371 to 930 for salicylate and 2531 to 1591 for cimetidineData analyses were carried out using LabSolutions (ver 580Shimadzu) and quantified using sample peak height

28 Statistical Analysis 119875 values were calculated using two-tailed Studentrsquos t-test for pairwise comparisons and one-wayanalysis of variance (ANOVA) followed by Bonferronirsquos testfor multiple comparisons unless otherwise stated A 119875 valueof lt005 was considered statistically significant Data areexpressed as mean plusmn SEM

3 Results

31 Diabetic Conditions The nonfasting blood glucose levelswere 500sim600mgdL in diabetic mice at 4 to 16 weeks ofage (Figure 1(a)) representing a sustained hyperglycemiaInsulin treatment twice a day significantly reduced hyper-glycemia after the injection of the long-acting insulin analog(Figure 1(a)) Serious hypoglycemic troubles including thedeath of the mice were not seen in this study Salicylate andpyridoxamine treatments in diabetic mice did not result instatistically significant decreases in nonfasting blood glucoselevels but there was a tendency of lowering the glucoselevels in the salicylate treatment group (Figure 1(a)) Thebody weight (BW) of the diabetic groups was significantlylower than that of nondiabetic control but no statisticallysignificant differences were observed among diabetic groupswith or without treatment at any time points during theobservation period (Table 2 and data not shown) Foodintake was significantly increased in diabetic mice whencompared to nondiabetic controls (Table 2) Insulin treat-ment negated the increased food intake in diabetic micesalicylate treatment also had a partial inhibitory effect onfeeding behavior (Table 2) Pyridoxamine did not result inchanges in food intake (Table 2)The same trends were noted


4 8 12 16Weeks of age

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ood

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ey w

eigh

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(b)

Figure 1 Blood glucose levels and nephromegaly Nonfasting blood glucose levels in nondiabetic control (Control) diabetic mice withouttreatment (DM) diabetic mice with insulin treatment (subcutaneous injection of Levemir 2 unitskg body weight twice a day) (DM + Ins)diabetic mice with salicylate treatment (sodium salicylate 3 gkg in the diets) (DM + Sal) and diabetic mice with pyridoxamine treatment(pyridoxamine dihydrochloride 1 gL in the drinking water) (DM+ Pyrid) (a) Kidney weight per body weight ratio (gg) (b) Control (119899 = 8)DM (119899 = 19) DM + Ins (119899 = 8) DM + Sal (119899 = 12) and DM + Pyrid (119899 = 15) Values are mean plusmn SEM

Table 2 Diabetic parameters in 16-week-old diabetic mice with or without treatment

Control DM DM + Ins DM + Sal DM + PyridBW (g) 577 plusmn 15 462 plusmn 11lowast 410 plusmn 15lowast 404 plusmn 10lowast 401 plusmn 09lowast

Food intake (gd) 458 plusmn 008 680 plusmn 006lowast 395 plusmn 003 569 plusmn 013lowast 643 plusmn 012lowast

Heart (g) 0195 plusmn 0005 0204 plusmn 0007 0191 plusmn 0010 0182 plusmn 0004 0184 plusmn 0005lowastP lt 005 versus Control data are expressed as mean plusmn SEM

in water drinking during the observation periods nondia-betic control 594 plusmn 0281mLmouseday diabetes withouttreatment 1968plusmn0454mLmouseday diabetes with insulintreatment 426 plusmn 0090mLmouseday diabetes with salicy-late treatment 1335 plusmn 0370mLmouseday diabetes withpyridoxamine treatment 172 plusmn 0776mLmouseday

32 Kidney Function and Histological Findings After 16weeks of treatment functional and macro- and microscopicanalyses of the kidneys were performed The enlargement ofthe kidney expressed as kidney weight per BW was notedin diabetic mice (Figure 1(b)) Insulin treatment significantlyameliorated this renal size change but treatment with sal-icylate or pyridoxamine did not (Figure 1(b)) In contrastheart weight was not so different among all groups (Table 2)Albuminuria was assessed by the urinary albumin-creatinineratio (ACR) in the animals at 20 weeks of age Diabetic micewithout treatment showed significant elevation of the ACRwhen compared to nondiabetic control mice (Figure 2(a))All the treatment groups given insulin salicylate or pyridox-amine were found to significantly decrease the albuminuriaseen in diabetic mice (Figure 2(a)) In this study salicylate(3 gkg diet) did not induce tubular damages such as high

excretion of urinary beta-2 microglobulin We next assessedserum creatinine levels in the mice which reflect filtrationrate by the kidneys The mouse serum creatinine levels indiabetes without treatment were significantly higher thanthose in nondiabetic controls at 16 weeks of age (Figure 2(b))Treatment with insulin salicylate or pyridoxamine signifi-cantly attenuated the increased serum creatinine levels seenin diabetic mice (Figure 2(b))

Next histopathological analyses were performed Theperiodic acid-Schiff (PAS) staining of the kidney tissuesections is shown in Figure 3 Accumulation of PAS-positivematerials in the glomeruli was prominent in the kidneyfrom diabetic mice without treatment when compared tonondiabetic controls Either insulin salicylate or pyridoxam-ine treatment attenuated the deposition of the PAS-positivematerials in the glomeruli (Figure 3) Quantitative evaluationof the histological indices was then performed There wereno significant differences in glomerular area among thegroups (Figure 4(a)) However the tuft area of the diabeticgroup without treatment was significantly larger than that ofnondiabetic controls and the increased tuft area was signif-icantly attenuated by the treatment with insulin salicylateor pyridoxamine in diabetic mice (Figure 4(b)) Glomerular


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Figure 2 Albuminuria and serum creatinine levels Albuminuria was evaluated as urinary albumin-creatinine ratio (ACR) in nondiabeticcontrol (Control) diabetic mice without treatment (DM) diabetic mice with insulin treatment (DM + Ins) diabetic mice with salicylatetreatment (DM + Sal) and diabetic mice with pyridoxamine treatment (DM + Pyrid) (a) Serum creatinine levels (mgdL) (b) Control(119899 = 8) DM (119899 = 19) DM + Ins (119899 = 8) DM + Sal (119899 = 12) and DM + Pyrid (119899 = 15) Values are mean plusmn SEM

cell number was counted and the glomerular cell numberper tuft area was determined This index was found to beincreased under the diabetic condition (Figure 4(c)) and thisupregulation was prevented by the treatment with insulinsalicylate or pyridoxamine (Figure 4(c)) Glomerulosclerosisat 20 weeks of age was evaluated and expressed as sclerosisindex Sclerosis increased under diabetic conditions andwas significantly prevented by the treatment with insulinsalicylate or pyridoxamine (Figure 4(d))

33 Macrophage Infiltration in Kidneys We next focusedon macrophage infiltration and accumulation evaluatedby flow cytometry Macrophages were defined asPIminusCD45+CD11b+F480+ cells (Figure 5(a)) Although totalliving cell number in the whole kidney was invariant amongeach group (data not shown) macrophage in the kidneyand the total number of the macrophages were significantlyincreased under the diabetic condition and the increasewas significantly blocked by the treatment with salicylate(Figures 5(b) and 5(c)) The tendency of downregulation inmacrophage accumulation was observed in the groups withinsulin or pyridoxamine treatment (Figures 5(b) and 5(c))

34 Gene Expression Analyses of Kidneys Inflammatory andfibrotic gene expressions were next examined Expression ofglyoxalase 1 (Glo1) mRNA which catalyze cytotoxic methyl-glyoxal a highly active glycating agent was significantlydownregulated in kidneys from diabetic mice (Figure 6)

Only salicylate treatment slightly but significantly upregu-lated Glo1 mRNA (Figure 6) Toll-like receptor 4 (Tlr4) geneexpression was not altered among the groups (Figure 6)Gene expression patterns were similar for interleukin (Il)1120573Il10 vascular cell adhesion molecule- (Vcam-) 1 monocytechemoattractant protein- (Mcp-) 1 Mcp-2 C-C chemokinereceptor type 2 (Ccr2) and collagen 1a1 (Col1a1) (Figure 6)The diabetic condition unregulated these mRNA levels incomparison with the nondiabetic sit and salicylate andpyridoxamine treatments downregulated the increase thatwas seen in the hyperglycemic condition (Figure 6)

4 Discussion

In this study we have found preventive effects of salicy-late and pyridoxamine on early stage indices of diabeticnephropathy including albuminuria and enlarged tuft areaas well as late stage kidney injuries such as an increasein serum creatinine and glomerulosclerosis Salicylate wasfound to inhibit macrophage infiltration into the kidneys andto downregulate gene expressions of Il1120573 Mcp-1 Ccr2 andCol1a1 showing anti-inflammatory and antifibrotic actionsagainst diabetic kidney injury Also salicylate could sig-nificantly upregulate the antiglycating enzyme Glo1 mRNAlevels The anti-inflammatory gene Il10 was upregulated dueto compensation in diabetic mice and downregulated aftertreatment with pyridoxamine or salicylate On the otherhand pyridoxamine did not have a significant inhibitory


Control DM

DM + Ins DM + Sal

DM + Pyrid

Figure 3 Periodic acid-Schiff (PAS) stain of kidneys Nondiabetic control (Control) diabetic mice without treatment (DM) diabetic micewith insulin treatment (DM + Ins) diabetic mice with salicylate treatment (DM + Sal) and diabetic mice with pyridoxamine treatment (DM+ Pyrid) Bar indicates 50120583m

action on macrophage infiltration but demonstrated down-regulation of inflammatory and fibrotic gene expressionssuch as Il1120573 Mcp-1 Mcp-2 Ccr2 and Col1a1 In the presentstudy we isolated total RNAs from mouse whole kidneys forthe analyses of various gene expressions However becausethe extracted RNAs and the resultant data were derived frommore than 90 of tubularinterstitial components it is likelythat the present findings may be limited and confoundingfor interpreting glomerular changes Further histologicalexaminationsmay help to understand the expressions of thesemolecules in specific cell types of the kidneys

Salicylates are among the most commonly used non-steroidal anti-inflammatory drugs and nonacetylated (egsodium salicylate salsalate and trilisate) and acetylated(aspirin) forms of salicylate are widely used to reduce feverpain and inflammation that occur with conditions such asrheumatic arthritis Even at low doses aspirin effectivelyinhibits cyclooxygenase enzymes COX 1 and 2 through

covalent transacetylation of active site serine residues [21]Nonacetylated salicylates are reported not tomodify activitiesof cyclooxygenase enzymes [22] At a higher concentrationboth nonacetylated salicylates and aspirin are known toinhibit the IKK120573NF-120581B axis an important regulator ofinflammation [23 24] Aspirin is reported to inhibit glycoxi-dation and AGE-cross-link formation [25] but not glycationitself In a clinical study treatment with salsalate a nonacety-lated salicylate resulted in noninhibitory effects on AGEformation [18] In this study we used a high dose of sodiumsalicylate at 3 gkg diet resulting in the serum concentrationat 1026 plusmn 46 120583M corresponding to the dose for humantherapy and could see inhibition of the development ofdiabetic nephropathy in mice by nonacetylated sodium sal-icylate A previous study demonstrates that obesity activatesthe IKK120573NF-120581B pathway in animals and that inhibition ofthis pathway by salicylates improves obesity-induced diabetes[22 26] In addition clinical studies show that salicylates


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Figure 4 Histological evaluations of kidneys Glomerular area (a) tuft area (b) glomerular cell number (c) and sclerosis indices (d) areshown Nondiabetic control (Control) (119899 = 8) diabetic mice without treatment (DM) (119899 = 19) diabetic mice with insulin treatment (DM+ Ins) (119899 = 8) diabetic mice with salicylate treatment (DM + Sal) (119899 = 12) and diabetic mice with pyridoxamine treatment (DM + Pyrid)(119899 = 15) Values are mean plusmn SEM

halve the NF-120581B activity in circulating immune cells [27]An animal study showed that salicylates induced a 50decrease in the number of Ly6Chi monocytes which expressCCR2 mediating the migration of monocytes into localinflammatory sites [28] This is compatible with our findingsin this studyMoreover it is interesting that salicylate induced

the gene expression of Glo1 which catalyzes the conversionof cytotoxic methylglyoxal to S-D-lactoylglutathione and D-lactate which suppresses glycation-mediated cellular damageassociated with diabetes and aging

Pyridoxamine a vitamin B6 is reported to have manyeffects including (1) inhibition of AGE formation by trapping


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ey

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Figure 5 Flow cytometric data of kidneys Macrophages were defined as PIminusCD45+CD11b+F480+ cells in the kidney The final gating panelof CD11b- and F480-positive cell cluster is shown (a) Macrophage in the kidney (b) and the total number of the macrophages (c) werecalculated Nondiabetic control (Control) (119899 = 8) diabetic mice without treatment (DM) (119899 = 19) diabetic mice with insulin treatment (DM+ Ins) (119899 = 8) diabetic mice with salicylate treatment (DM + Sal) (119899 = 12) and diabetic mice with pyridoxamine treatment (DM + Pyrid)(119899 = 15) Values are mean plusmn SEM

dicarbonyl intermediates during glycation reaction (2) scav-enging toxic carbonyl products of glucose and lipid degra-dation and (3) trapping of reactive oxygen species (ROS)[12 29] The dosage of pyridoxamine (sim200mgkgday) usedin this study achieving the serum concentrations at 042 plusmn029 120583M was within a less toxic range and its preclinicalefficacy has been proven in other animal models of earlydiabetic nephropathy such as KK-AyTa and streptozotocin-induced diabetic rats [13 14] The serum concentration ofpyridoxamine was lower than our expectations and previousreports [30] this may be due to its instability in aqueous solu-tions and photosensitivity as well as different administration

methods Using our mouse model of diabetic nephropathypyridoxamine treatment with 200mgkgday significantlyimproved early to late stages of kidney injuries

In conclusion this study demonstrated that an anti-inflammatory reagent salicylate as well as an antiglycoox-idative drug pyridoxamine could inhibit the developmentof diabetic nephropathy in mice Both drugs significantlyblocked diabetes-induced inflammatory and profibrotic geneexpressions Attenuation of macrophage infiltration into thekidneys and upregulation of Glo1 mRNA expression werefound only in the salicylate treatment group Synergisticand additive effects of the combination with salicylate and


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DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

0

20

40

60

80

100

120

140Gl

o1T

bp

0005

01015

02025

03035

Tlr4

Tbp

0005

01015

02025

03035

04045

Il1120573

Tbp

00005

0010015

0020025

0030035

004

Il10

Tbp

0

005

01

015

02

025

03

035

Vcam

1Tb

p

00005

0010015

0020025

0030035

0040045

Col1

a1 T

bp

0

005

01

015

02

025

03

Ccr2

Tbp

0

05

1

15

2

25

3

Mcp

-2 T

bp

0005

01015

02025

03035

04

Mcp

-1T

bplowast lowast

lowast lowast

lowast lowast lowast lowast lowast lowast lowastlowastlowast lowast

lowast lowast lowast


Figure 6 Gene expressions of kidneys The mRNA expression levels were measured by qRT-PCR and normalized to TATA-binding protein(Tbp) Following primer sets were used for the detection (Table 1) glyoxalase 1 (Glo1) toll-like receptor 4 (Tlr4) interleukin (Il)1b Il10vascular cell adhesion molecule- (Vcam-) 1 monocyte chemoattractant protein- (MCP-) 1 Mcp-2 C-C chemokine receptor type 2 (Ccr2)and collagen 1a1 (Col1a1) Nondiabetic control (Control) (119899 = 8) diabetic mice without treatment (DM) (119899 = 19) diabetic mice with insulintreatment (DM + Ins) (119899 = 8) diabetic mice with salicylate treatment (DM + Sal) (119899 = 12) and diabetic mice with pyridoxamine treatment(DM + Pyrid) (119899 = 15) Values are mean plusmn SEM

pyridoxamine as well as therapeutic effects of these drugswill hopefully be investigated in future studies Interventionalstrategies using these drugs may be useful for diabeticnephropathy

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

The authors want to thank Ms Yuko Niimura for herassistance and acknowledge financial support from Grants-in-Aid for Scientific Research from the Japan Society forPromotion of Science (24590375 25461335 and 26450152)

References

[1] J L Gross M J de Azevedo S P Silveiro L H CananiM L Caramori and T Zelmanovitz ldquoDiabetic nephropathydiagnosis prevention and treatmentrdquoDiabetes Care vol 28 no1 pp 164ndash176 2005

[2] L Gnudi S M Thomas and G Viberti ldquoMechanical forcesin diabetic kidney disease a trigger for impaired glucosemetabolismrdquo Journal of the American Society of Nephrology vol18 no 8 pp 2226ndash2232 2007

[3] H Abe T Matsubara H Arai and T Doi ldquoRole of Smad1 indiabetic nephropathy molecular mechanisms and implicationsas a diagnostic markerrdquo Histology and Histopathology vol 26no 4 pp 531ndash541 2011

[4] M Brownlee ldquoBiochemistry and molecular cell biology ofdiabetic complicationsrdquo Nature vol 414 no 6865 pp 813ndash8202001

[5] J M Forbes M E Cooper M D Oldfield and M CThomas ldquoRole of advanced glycation end products in diabeticnephropathyrdquo Journal of theAmerican Society ofNephrology vol14 no 3 pp S254ndashS258 2003

[6] H Yamamoto T Watanabe Y Yamamoto et al ldquoRAGE indiabetic nephropathyrdquo Current Molecular Medicine vol 7 no8 pp 752ndash757 2007

[7] Y Yamamoto and H Yamamoto ldquoReceptor for advancedglycation end-products-mediated inflammation and diabeticvascular complicationsrdquo Journal of Diabetes Investigation vol2 no 3 pp 155ndash157 2011


[8] Y Yamamoto I Kato T Doi et al ldquoDevelopment and pre-vention of advanced diabetic nephropathy in RAGE-over-expressing micerdquo Journal of Clinical Investigation vol 108 no2 pp 261ndash268 2001

[9] K-M Myint Y Yamamoto T Doi et al ldquoRAGE control ofdiabetic nephropathy in a mouse model effects of RAGEgene disruption and administration of low-molecular weightheparinrdquo Diabetes vol 55 no 9 pp 2510ndash2522 2006

[10] Y Yamamoto and H Yamamoto ldquoControlling the receptor foradvanced glycation end-products to conquer diabetic vascularcomplicationsrdquo Journal of Diabetes Investigation vol 3 no 2 pp107ndash114 2012

[11] M T T Win Y Yamamoto S Munesue et al ldquoRegulation ofRAGE for attenuating progression of diabetic vascular compli-cationsrdquo Experimental Diabetes Research vol 2012 Article ID894605 8 pages 2012

[12] A A Booth R G Khalifah and B G Hudson ldquoThiaminepyrophosphate and pyridoxamine inhibit the formation ofantigenic advanced glycation end-products comparison withaminoguanidinerdquo Biochemical and Biophysical Research Com-munications vol 220 no 1 pp 113ndash119 1996

[13] T P Degenhardt N L Alderson D D Arrington et alldquoPyridoxamine inhibits early renal disease and dyslipidemia inthe streptozotocin-diabetic ratrdquo Kidney International vol 61no 3 pp 939ndash950 2002

[14] N L Alderson M E Chachich N N Youssef et al ldquoTheAGE inhibitor pyridoxamine inhibits lipemia and developmentof renal and vascular disease in Zucker obese ratsrdquo KidneyInternational vol 63 no 6 pp 2123ndash2133 2003

[15] M-J Yin Y Yamamoto and R B Gaynor ldquoThe anti-inflammatory agents aspirin and salicylate inhibit the activityof I120581B kinase-120573rdquo Nature vol 396 no 6706 pp 77ndash80 1998

[16] A B Goldfine V Fonseca K A Jablonski L Pyle M A Statenand S E Shoelson ldquoThe effects of salsalate on glycemic controlin patients with type 2 diabetes a randomized trialrdquo Annals ofInternal Medicine vol 152 no 6 pp 346ndash357 2010

[17] A B Goldfine V Fonseca K A Jablonski et al ldquoTargetingInflammation Using Salsalate in Type 2 Diabetes Study TeamSalicylate (salsalate) in patients with type 2 diabetes a random-ized trialrdquo Annals of Internal Medicine vol 159 no 1 pp 1ndash122013

[18] J I Barzilay K A Jablonski V Fonseca et al ldquoThe impact ofsalsalate treatment on serum levels of advanced glycation endproducts in type 2 diabetesrdquo Diabetes Care vol 37 no 4 pp1083ndash1091 2014

[19] T Takamura I Kato N Kimura et al ldquoTransgenic miceoverexpressing type 2 nitric-oxide synthase in pancreatic 120573 cellsdevelop insulin-dependent diabetes without insulitisrdquo Journalof Biological Chemistry vol 273 no 5 pp 2493ndash2496 1998

[20] S Ohashi H Abe T Takahashi et al ldquoAdvanced glycation endproducts increase collagen-specific chaperone protein inmousediabetic nephropathyrdquoThe Journal of Biological Chemistry vol279 no 19 pp 19816ndash19823 2004

[21] P Patrignani S Tacconelli E Piazuelo et al ldquoReappraisal ofthe clinical pharmacology of low-dose aspirin by comparingnovel direct and traditional indirect biomarkers of drug actionrdquoJournal of Thrombosis and Haemostasis vol 12 no 8 pp 1320ndash1330 2014

[22] M Yuan N Konstantopoulos J Lee et al ldquoReversal of obesity-and diet-induced insulin resistance with salicylates or targeteddisruption of Ikk120573rdquo Science vol 293 no 5535 pp 1673ndash16772001

[23] S E Shoelson J Lee and M Yuan ldquoInflammation and theIKK120573I120581BNF-120581B axis in obesity- and diet-induced insulinresistancerdquo International Journal of Obesity vol 27 pp S49ndashS522003

[24] Y T Kwak J Guo J Shen and R B Gaynor ldquoAnalysis ofdomains in the IKK120572 and IKK120573 proteins that regulate theirkinase activityrdquo Journal of Biological Chemistry vol 275 no 19pp 14752ndash14759 2000

[25] E C Abraham M S Swamy and R E Perry ldquoNonenzymaticglycosylation (glycation) of lens crystallins in diabetes andagingrdquo Progress in Clinical and Biological Research vol 304 pp123ndash139 1989

[26] D Cai M Yuan D F Frantz et al ldquoLocal and systemic insulinresistance resulting from hepatic activation of IKK-120573 and NF-120581Brdquo Nature Medicine vol 11 no 2 pp 183ndash190 2005

[27] A B Goldfine R Silver W Aldhahi et al ldquoUse of salsalate totarget inflammation in the treatment of insulin resistance andtype 2 diabetesrdquo Clinical and Translational Science vol 1 no 1pp 36ndash43 2008

[28] M-S Kim Y Yamamoto K Kim et al ldquoRegulation of diet-induced adipose tissue and systemic inflammation by salicylatesand Pioglitazonerdquo PLoS ONE vol 8 no 12 Article ID e828472013

[29] P A Voziyan and B G Hudson ldquoPyridoxamine as a multi-functional pharmaceutical targeting pathogenic glycation andoxidative damagerdquo Cellular and Molecular Life Sciences vol 62no 15 pp 1671ndash1681 2005

[30] Y Mori T Kakuta T Miyakogawa et al ldquoEffect of Scavengingcirculating reactive carbonyls by oral pyridoxamine in uremicrats on peritoneal dialysisrdquo Therapeutic Apheresis and Dialysis2016

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


progressive glomerulosclerosis with renal dysfunction com-pared to diabetic littermates lacking the RAGE transgene [8]Moreover homozygous RAGE-null diabetic mice failed todevelop mesangial matrix expansion or glomerulosclerosis[9] Intracellular RAGE signaling involves activation of tran-scriptional factor NF120581B activity thus enhancing proinflam-matory reactions [10 11]

To prevent and treat diabetic kidney disease we examinedthe effects of pyridoxamine an antiglycating drug andsalicylate an anti-inflammatory drug on the developmentof diabetic nephropathy using our unique animal modelPyridoxamine is one of the natural forms of vitamin B6 and isknown to act as a nucleophilic trap for reactive carbonyl inter-mediates in AGE formation [12] Pyridoxamine is reportedto prevent the development of early stage nephropathy in arat model of diabetes [13 14] The effectiveness on late stagediabetic nephropathy including glomerulosclerosis is not yetfully elucidated in a rat model of diabetes [13] Salicylate is awell-known anti-inflammatory compound that inhibits I120581Bkinase-120573 (IKK120573) and is downstream of NF120581B [15] Recentclinical studies have shown that salsalate a nonacetylatedsalicylate lowered fasting blood glucose levels and HbA1c aswell as markers of inflammation [16 17] but did not decreasethe formation late glycation products [18]

In this study we studied the effects of salicylate comparedto pyridoxamine on early to late stages of diabetic nephropa-thy in our original mouse model of diabetic kidney injurieswith insulin treatment group as a control

2 Materials and Methods

21 Animals The male mice of a type 1 diabetic mouseline that overexpresses inducible NO synthase (iNOS) inpancreatic 120573-cells and control CD1 (ICR) CD-1 background(Charles River Japan) were used in this study [8 19]They were fed a high-calorie diet (Labo H standard NosanYokohma Japan) that contained 311 protein 82 lipids33 fiber 63 ash and 81 water The transgenic miceconsistently develop overt diabetes as early as 1 week afterbirth due to the NO-mediated selective destruction of insulinproducing pancreatic 120573-cells which results in advanceddiabetic nephropathy [8 19 20] Animals were treated inaccordance with the Fundamental Guidelines for ProperConducts of Animal Experiment and Related Activities inAcademic Research Institutions under the jurisdiction ofthe Ministry of Education Culture Sports Science andTechnology of Japan Animal experiments were approvedby the Committee on Animal Experimentation of KanazawaUniversity

22 Animal Groups with Different Treatment Pyridoxamine-dihydrochloride-monohydrate (4-aminomethyl-3-hydroxy-2-methyl-5-oxymethylpyridinedihydrochloride) was pur-chased from Tokyo Chemical Industry Co Japan Thepyridoxamine dihydrochloride was given at 1 gL (sim200mgkgday) in the drinking water from 4 to 20 weeks of age for16 weeks resulting in the serum concentration at 042 120583M(lt003 120583M in nontreated mice) Salicylate (sodium salicylate

Sigma-Aldrich Japan) was given at 3 gkg in their dietsyielding the serum concentration at 1026 120583M (not detectedin nontreated mice) For the insulin treatment group along-acting insulin analog of insulin detemir (Levemir NovoNordisk) was subcutaneously injected twice a day at the doseof 2 unitskg body weight

The following five animal groups were subjected tophenotypic analyses (1) nondiabetic control (119899 = 8) (2)diabetic mice without treatment (119899 = 19) (3) diabetic micewith insulin treatment (119899 = 8) (4) diabetic mice withsalicylate treatment (119899 = 12) and (5) diabetic mice withpyridoxamine treatment (119899 = 15)

23 Biochemical Assays Urinary samples were collectedby manual manipulation then kept in Eppendorf tubes atminus80∘C Blood glucose levels were determined using Glu-cocard (Arkray Japan) Body weight (BW) and nonfastingblood glucose levels were checked at 4 8 12 16 and 20weeks of age Serum and urinary creatinine levels weremonitored at 12 16 and 20 weeks of age The urinaryalbumin-creatinine ratio (ACR) was calculated at 20 weeksof age Serum and urinary creatinine were measured usingthe creatinine-test (Wako Osaka Japan) Urinary albuminand beta-2 microglobulin levels were measured by a mouseAlbumin ELISA Quantitation kit (AlbuWell II M ExocellPhiladelphia PA) and a mouse BMG1205732-MG ELISA kit(Elabscience Japan) respectively

24 Histopathology After sacrificing the animals at 20 weeksof age the left kidney was obtained and fixed in a Carnoyrsquossolution (10 acetic acid 60methanol and 3 chloroform)or 4 paraformaldehyde For 48 hr fixation paraformalde-hyde and Carnoyrsquos solutions were replaced by phosphatebuffer saline (PBS) and 70 methanol respectively Thekidney specimens were sectioned at 1 120583m thicknesses fol-lowed by the periodic acid-Schiff (PAS) periodic acid-methenamine silver (PAM) or hematoxylin and eosin (HampE)stain and imaged using a lightmicroscope (ZEISSmicroscopewith Fujifilm digital camera HC 300Z) Quantitative exami-nations were done on at least 50 glomeruli per mouse Theseverity of the renal sclerosis was scored by multiple analystson an arbitrary scale from 0 to 4 [8 9] The mean glomerulararea was determined as described previously [8 9]

25 Flow Cytometry (FACS) To evaluate macrophage infil-tration cells from the entire right kidney from each animalgroup at 20 weeks of age were subjected to FACS analysisThe kidney was cut into small pieces by scissors and digestedby incubating with collagenase II (1 120583gmL) Cells werethen washed and resuspended in staining buffer (PBS with2 FCS) containing FcBlock (BD Biosciences) Cells werestained by incubating for 15min at 4∘C in the dark with thefollowing antibodies CD45-PE (eBioscience) CD11bndashAPC-Cy7 (BD Biosciences) and F480-PE-Cy7 (BD Biosciences)Cells were resuspended in 200120583L of staining buffer con-taining 02120583gmLpropidium iodide (Sigma-Aldrich) filteredthrough a 100 120583m mesh and analyzed by FACSAria II (BDBiosciences) To quantify viable cell numbers liquid counting
















18(21 times




3 Results




0

100

200

300

400

500

600

700Bl

ood

gluc

ose (

mg

dL)

ControlDMDM + Ins

DM + SalDM + Pyrid

(a)

Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

0

0002

0004

0006

0008

001

0012

0014

Kidn

ey w

eigh

tBW

ratio

lowastlowast

lowastP lt 005

(b)











Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

0

02

04

06

08

1

12

14

16

18U

rinar

y al

bum

inc

reat

inin

e rat

io



(a)

Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

0

02

04

06

08

1

12

14

16

Seru

m cr

eatin

ine (

mg

dL)



(b)






4 Discussion



Control DM

DM + Ins DM + Sal

DM + Pyrid






Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

0

1

2

3

4

5

6

7G

lom

erul

ar ar

ea (times

10minus4120583

m2)

(a)

Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid


0

05

1

15

2

25

3

35

4

Tuft

area

(times10

minus4120583

m2)


(b)

Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

0

5

10

15

20

25

30

35

40

45

Glo

mer

ular

cell

num

ber

lowastlowast

lowastlowast



(c)

Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

0

05

1

15

2

25

3

Scle

rosis


lowastlowast


(d)






0

CD11b

F48

0

Macrophages

0 101 102 103

101

102

103

(a)

Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

0

02

04

06

08

1

12

14

Mac

roph

ages

()

P lt 005

(b)

Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

P lt 005

0

10000

20000

30000

40000

50000

60000

70000

80000

90000M

acro

phag

e num

ber

kidn

ey

(c)






Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

0

20

40

60

80

100

120

140Gl

o1T

bp

0005

01015

02025

03035

Tlr4

Tbp

0005

01015

02025

03035

04045

Il1120573

Tbp

00005

0010015

0020025

0030035

004

Il10

Tbp

0

005

01

015

02

025

03

035

Vcam

1Tb

p

00005

0010015

0020025

0030035

0040045

Col1

a1 T

bp

0

005

01

015

02

025

03

Ccr2

Tbp

0

05

1

15

2

25

3

Mcp

-2 T

bp

0005

01015

02025

03035

04

Mcp

-1T

bplowast lowast

lowast lowast






Competing Interests


Acknowledgments


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of































18(21 times




3 Results




0

100

200

300

400

500

600

700Bl

ood

gluc

ose (

mg

dL)

ControlDMDM + Ins

DM + SalDM + Pyrid

(a)

Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

0

0002

0004

0006

0008

001

0012

0014

Kidn

ey w

eigh

tBW

ratio

lowastlowast

lowastP lt 005

(b)











Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

0

02

04

06

08

1

12

14

16

18U

rinar

y al

bum

inc

reat

inin

e rat

io



(a)

Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

0

02

04

06

08

1

12

14

16

Seru

m cr

eatin

ine (

mg

dL)



(b)






4 Discussion



Control DM

DM + Ins DM + Sal

DM + Pyrid






Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

0

1

2

3

4

5

6

7G

lom

erul

ar ar

ea (times

10minus4120583

m2)

(a)

Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid


0

05

1

15

2

25

3

35

4

Tuft

area

(times10

minus4120583

m2)


(b)

Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

0

5

10

15

20

25

30

35

40

45

Glo

mer

ular

cell

num

ber

lowastlowast

lowastlowast



(c)

Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

0

05

1

15

2

25

3

Scle

rosis


lowastlowast


(d)






0

CD11b

F48

0

Macrophages

0 101 102 103

101

102

103

(a)

Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

0

02

04

06

08

1

12

14

Mac

roph

ages

()

P lt 005

(b)

Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

P lt 005

0

10000

20000

30000

40000

50000

60000

70000

80000

90000M

acro

phag

e num

ber

kidn

ey

(c)






Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

0

20

40

60

80

100

120

140Gl

o1T

bp

0005

01015

02025

03035

Tlr4

Tbp

0005

01015

02025

03035

04045

Il1120573

Tbp

00005

0010015

0020025

0030035

004

Il10

Tbp

0

005

01

015

02

025

03

035

Vcam

1Tb

p

00005

0010015

0020025

0030035

0040045

Col1

a1 T

bp

0

005

01

015

02

025

03

Ccr2

Tbp

0

05

1

15

2

25

3

Mcp

-2 T

bp

0005

01015

02025

03035

04

Mcp

-1T

bplowast lowast

lowast lowast






Competing Interests


Acknowledgments


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















0

100

200

300

400

500

600

700Bl

ood

gluc

ose (

mg

dL)

ControlDMDM + Ins

DM + SalDM + Pyrid

(a)

Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

0

0002

0004

0006

0008

001

0012

0014

Kidn

ey w

eigh

tBW

ratio

lowastlowast

lowastP lt 005

(b)











Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

0

02

04

06

08

1

12

14

16

18U

rinar

y al

bum

inc

reat

inin

e rat

io



(a)

Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

0

02

04

06

08

1

12

14

16

Seru

m cr

eatin

ine (

mg

dL)



(b)






4 Discussion



Control DM

DM + Ins DM + Sal

DM + Pyrid






Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

0

1

2

3

4

5

6

7G

lom

erul

ar ar

ea (times

10minus4120583

m2)

(a)

Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid


0

05

1

15

2

25

3

35

4

Tuft

area

(times10

minus4120583

m2)


(b)

Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

0

5

10

15

20

25

30

35

40

45

Glo

mer

ular

cell

num

ber

lowastlowast

lowastlowast



(c)

Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

0

05

1

15

2

25

3

Scle

rosis


lowastlowast


(d)






0

CD11b

F48

0

Macrophages

0 101 102 103

101

102

103

(a)

Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

0

02

04

06

08

1

12

14

Mac

roph

ages

()

P lt 005

(b)

Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

P lt 005

0

10000

20000

30000

40000

50000

60000

70000

80000

90000M

acro

phag

e num

ber

kidn

ey

(c)






Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

0

20

40

60

80

100

120

140Gl

o1T

bp

0005

01015

02025

03035

Tlr4

Tbp

0005

01015

02025

03035

04045

Il1120573

Tbp

00005

0010015

0020025

0030035

004

Il10

Tbp

0

005

01

015

02

025

03

035

Vcam

1Tb

p

00005

0010015

0020025

0030035

0040045

Col1

a1 T

bp

0

005

01

015

02

025

03

Ccr2

Tbp

0

05

1

15

2

25

3

Mcp

-2 T

bp

0005

01015

02025

03035

04

Mcp

-1T

bplowast lowast

lowast lowast






Competing Interests


Acknowledgments


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

0

02

04

06

08

1

12

14

16

18U

rinar

y al

bum

inc

reat

inin

e rat

io



(a)

Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

0

02

04

06

08

1

12

14

16

Seru

m cr

eatin

ine (

mg

dL)



(b)






4 Discussion



Control DM

DM + Ins DM + Sal

DM + Pyrid






Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

0

1

2

3

4

5

6

7G

lom

erul

ar ar

ea (times

10minus4120583

m2)

(a)

Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid


0

05

1

15

2

25

3

35

4

Tuft

area

(times10

minus4120583

m2)


(b)

Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

0

5

10

15

20

25

30

35

40

45

Glo

mer

ular

cell

num

ber

lowastlowast

lowastlowast



(c)

Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

0

05

1

15

2

25

3

Scle

rosis


lowastlowast


(d)






0

CD11b

F48

0

Macrophages

0 101 102 103

101

102

103

(a)

Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

0

02

04

06

08

1

12

14

Mac

roph

ages

()

P lt 005

(b)

Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

P lt 005

0

10000

20000

30000

40000

50000

60000

70000

80000

90000M

acro

phag

e num

ber

kidn

ey

(c)






Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

0

20

40

60

80

100

120

140Gl

o1T

bp

0005

01015

02025

03035

Tlr4

Tbp

0005

01015

02025

03035

04045

Il1120573

Tbp

00005

0010015

0020025

0030035

004

Il10

Tbp

0

005

01

015

02

025

03

035

Vcam

1Tb

p

00005

0010015

0020025

0030035

0040045

Col1

a1 T

bp

0

005

01

015

02

025

03

Ccr2

Tbp

0

05

1

15

2

25

3

Mcp

-2 T

bp

0005

01015

02025

03035

04

Mcp

-1T

bplowast lowast

lowast lowast






Competing Interests


Acknowledgments


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Control DM

DM + Ins DM + Sal

DM + Pyrid






Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

0

1

2

3

4

5

6

7G

lom

erul

ar ar

ea (times

10minus4120583

m2)

(a)

Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid


0

05

1

15

2

25

3

35

4

Tuft

area

(times10

minus4120583

m2)


(b)

Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

0

5

10

15

20

25

30

35

40

45

Glo

mer

ular

cell

num

ber

lowastlowast

lowastlowast



(c)

Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

0

05

1

15

2

25

3

Scle

rosis


lowastlowast


(d)






0

CD11b

F48

0

Macrophages

0 101 102 103

101

102

103

(a)

Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

0

02

04

06

08

1

12

14

Mac

roph

ages

()

P lt 005

(b)

Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

P lt 005

0

10000

20000

30000

40000

50000

60000

70000

80000

90000M

acro

phag

e num

ber

kidn

ey

(c)






Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

0

20

40

60

80

100

120

140Gl

o1T

bp

0005

01015

02025

03035

Tlr4

Tbp

0005

01015

02025

03035

04045

Il1120573

Tbp

00005

0010015

0020025

0030035

004

Il10

Tbp

0

005

01

015

02

025

03

035

Vcam

1Tb

p

00005

0010015

0020025

0030035

0040045

Col1

a1 T

bp

0

005

01

015

02

025

03

Ccr2

Tbp

0

05

1

15

2

25

3

Mcp

-2 T

bp

0005

01015

02025

03035

04

Mcp

-1T

bplowast lowast

lowast lowast






Competing Interests


Acknowledgments


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

0

1

2

3

4

5

6

7G

lom

erul

ar ar

ea (times

10minus4120583

m2)

(a)

Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid


0

05

1

15

2

25

3

35

4

Tuft

area

(times10

minus4120583

m2)


(b)

Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

0

5

10

15

20

25

30

35

40

45

Glo

mer

ular

cell

num

ber

lowastlowast

lowastlowast



(c)

Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

0

05

1

15

2

25

3

Scle

rosis


lowastlowast


(d)






0

CD11b

F48

0

Macrophages

0 101 102 103

101

102

103

(a)

Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

0

02

04

06

08

1

12

14

Mac

roph

ages

()

P lt 005

(b)

Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

P lt 005

0

10000

20000

30000

40000

50000

60000

70000

80000

90000M

acro

phag

e num

ber

kidn

ey

(c)






Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

0

20

40

60

80

100

120

140Gl

o1T

bp

0005

01015

02025

03035

Tlr4

Tbp

0005

01015

02025

03035

04045

Il1120573

Tbp

00005

0010015

0020025

0030035

004

Il10

Tbp

0

005

01

015

02

025

03

035

Vcam

1Tb

p

00005

0010015

0020025

0030035

0040045

Col1

a1 T

bp

0

005

01

015

02

025

03

Ccr2

Tbp

0

05

1

15

2

25

3

Mcp

-2 T

bp

0005

01015

02025

03035

04

Mcp

-1T

bplowast lowast

lowast lowast






Competing Interests


Acknowledgments


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

CD11b

F48

0

Macrophages

0 101 102 103

101

102

103

(a)

Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

0

02

04

06

08

1

12

14

Mac

roph

ages

()

P lt 005

(b)

Con

trol

DM

DM

+ In

s

DM

+ S

al

DM

+ P

yrid

P lt 005

0

10000

20000

30000

40000

50000

60000

70000

80000

90000M

acro

phag

e num

ber

kidn

ey

(c)






Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

0

20

40

60

80

100

120

140Gl

o1T

bp

0005

01015

02025

03035

Tlr4

Tbp

0005

01015

02025

03035

04045

Il1120573

Tbp

00005

0010015

0020025

0030035

004

Il10

Tbp

0

005

01

015

02

025

03

035

Vcam

1Tb

p

00005

0010015

0020025

0030035

0040045

Col1

a1 T

bp

0

005

01

015

02

025

03

Ccr2

Tbp

0

05

1

15

2

25

3

Mcp

-2 T

bp

0005

01015

02025

03035

04

Mcp

-1T

bplowast lowast

lowast lowast






Competing Interests


Acknowledgments


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

DM

DM

+ In

sD

M +

Sal

DM

+ P

yrid

Con

trol

0

20

40

60

80

100

120

140Gl

o1T

bp

0005

01015

02025

03035

Tlr4

Tbp

0005

01015

02025

03035

04045

Il1120573

Tbp

00005

0010015

0020025

0030035

004

Il10

Tbp

0

005

01

015

02

025

03

035

Vcam

1Tb

p

00005

0010015

0020025

0030035

0040045

Col1

a1 T

bp

0

005

01

015

02

025

03

Ccr2

Tbp

0

05

1

15

2

25

3

Mcp

-2 T

bp

0005

01015

02025

03035

04

Mcp

-1T

bplowast lowast

lowast lowast






Competing Interests


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 preventive effect of salicylate and...

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