The Function And Mechanism Of The Treatment Of Peroxisome Proliferator Activated Receptors On Diabetic Nephropathy

BackgroundDiabetic nephropathy is a common complication in diabetes mellitus (DM)and chronic hyperglycemia is the primary mechanism mediating tissue pathology.Histological lesion of diabetic nephropathy is characterized by mesangial cell proliferation and extracellular matrix expansion.ECM plays important roles in the regulation of cell function,and its changes in composition and structure could have profound pathophysiological implications in the genesis of diabetic complications. In this context,mesangium matrix,which is the ECM found within the glomerulus,is of considerable interest because its accumulation correlates closely with renal impairment in diabetes.Regulation of mesangial matrix is a dynamic process involving synthetic as well as degradative processes.The latter involve a number of matrix metalloproteinases(MMPs).Many researches reported a reduced degradation as a possible mechanism leading to enlargement of Imbalanced msangium[. However,the exact effects of chronic hyperglycemia on matrix turnover and effective treatment on mesangium imbalancing in diabetic nephropathy has not been established.peroxisome proliferator-activated receptors(PPARs)are a family of nuclear receptors,Three PPAR isoforms,designated PPARα,PPARβ/δ,and PPARγ,have been cloned from xenopus laevis,mouse,rat,and humans. PPARs form heterodimers with the 9-cis retinoic acid receptor RXRα,which bind to characteristic DNA sequences termed peroxisome proliferator response elements(PPREs)located in the 59-flanking region of target genes and modulate the expression of genes involved in lipid b-oxidation,cell differentiation,and inflammation.PPARγligands, including the TZDs troglitazone,pioglitazone,and rosiglitazone. Accumulating evidence suggests that these drugs not only significantly improve insulin sensitivity but also may reduce microalbuminuria in diabetic nephropathy in genetically obese diabetic rodents and patients with typeⅡdiabetes.Troglitazone reduced expression of extracellular matrix proteins(fibronectin and typeⅠcollagen)and transforming growth factor-β.PPARr could suppress the expression of metalloproteinase 9 in human bronchial epithelial cells.These findings suggest that PPARγplays roles on extracellular matrix accumulation.Functional expression of PPARr has been reported in freshly isolated glomeruli and cultured mesangial cells.The aim of the first part of study was to evaluate the effect of PPARr on the extrscellular matrix turnover in human mesangial cell in vitro.At present PPARγligand has been widely applied in clinic,but this kind of medicines have some important side effect such as water retention which can cause the patient weight gain.Therefore the new clinical medication is urgently needed.At present studies have discovered nitro fatty acid is a kind of endogenous PPARγligand.And nitrooleate arouses the widespread interest because of its content rich, the structure simple,this research draws up to its pharmacological action,the second part of research was to evaluate the pharmacy function of nitrooleate on diabetic nephropathy.Methods1.the first part1.1 Mesangial cell culture The glomeruli were isolated from the normal kidney tissue which was separated from kidney tumor patient.The culture medium includes RPMI-1640 media(Hyclone,USA),20%fetal bovine serum(Hyclone,USA),20 mL/l penicillin-streptomycin(5000 IU/mL and 5000μg/mL,respectively),insulin(5μg/mL),transferrin(5μg/mL),and sodium selenite(5ng/mL,all came from Sigma,USA).The glomeruli were sieved through three film of stainless steel sieves,then grown in explant culture at 37℃,5%CO2.The mesangial cells were subsequently removed by trypsinization and reseeded into new bottles in the above medium for further growth and expansion after 14-16days.The mesangial cells were subsequently characterized by morphology method and immunohistochemical method:stellate shape,their ability to form hillocks,desmin(+)and keratin(-)in culture.1.2.RNA isolation and real-time quantitative reverse transcriptase PCR.Human mesangial cells(passages 3 to 7)were grown at a density of 1×105 cells per flask into flasks under 1640,after 3-4days,the medium was exchanged and the cells were cultured under starvation medium(1640 containing 0.5%FCS).24h after the cells were organized into three medium conditions:5 mM glucose(normal glucose group,NG),30 mM glucose(high glucose group,HG),5 mM glucose plus 25 mM mannitol (osmotic control,mannitol group,MG),or 30 mM glucose plus 3μM pioglitazone(pioglitazone treatment group,PIO)for 24h.To obtain total RNA from isolated mesangial cells,Trizol(Takara,Japan)was used according to the manufacturer's instruction.Total RNA was extracted from the supernatant and stored at -80℃.Complementary DNA(cDNA)was reversed transcripted using First Strand cDNA Synthesis Kit(AMV)(Takara, Japan)according to the manufacturer's instructions.The sequence of primers used for real-time PCR can be seen in tablel.Real-time PCR amplification was performed using the TaqMan Universal PCR Master Mix and the ABI Prism 7900 Sequence Detection System.PCR was performed for 40 cycles using 15 seconds denaturation step at 95℃,45 seconds annealing and extension step at 60-65℃.We usedβ-actin as a standard to assure equal synthesis of cDNA in different groups and the results were analyzed as ratios gene/β-actin and calculated threshold cycle numbers(CT)(i.e.,2-△△CT),according to the manufacturer's suggestions.1.3.Western blotting Nucluar protein was extracted by nuclear protein extraction kit(Pierce,USA)according to the manufacturer's suggestions.Total protein was extracted using method in published paper[21].The lysates were stored at -80℃until assayed.A bicinchoninic acid(BCA)protein assay(Pierce,USA)was used to quantify total protein in the medium according to developer's instructions,and the results of the assay were measured by absorbance at 562 nm using a microplate reader.An equal amount of the whole cell protein(40μg)was denatured at 60℃for 3 min,separated by SDS-PAGE,and transferred onto nitrocellulose membranes.The blots were blocked overnight with 5% nonfat dry milk in Tris-buffered saline(TBS),followed by incubation for 1 h with the PPARr polyclonal antibody(1:500,Santa cruz,USA),collagenⅣpolyclonal antibody(1:1000,Santa cruz,USA). After washing with TBS,blots were incubated with mouse anti-goat HRP-conjugated secondary antibody.Immune complexes were detected using ECL method and immunoreactive bands were quantified using Alphaimager 2200. Values were corrected with the absorbency of the internal control (actin).2.the second part2.1 using ZDF rats for therapeutic benefit for diabetic nephropathy.ZDF rats separated into two groups,each group included six rats.the treated group embedded mini pump subcutaneously,the rats were selected blood on each week during one month,after one month,the rats were killed,the kidney and heart were collected.2.2 using C57 mice for therapeutic benefit for diabetic nephropathy.The mice were anesthetized using isoflurane inhalation. After laparotomy,a 5-0 silk ligature was placed 1 cm from the cecal tip. The cecum was punctured twice with a 21-gauge needle and gently squeezed to express a 3 mm column of fecal material.The abdominal incision was closed in two layers with 6-0 nylon sutures.After surgery,1 ml of prewarmed normal saline was given intraperitoneally.All animals received a broad-spectrum antibiotic(imipenem/cilastatin;0.5mg per mice subcutaneously)in 1.5 ml of 3/4 normal saline at 6 h after surgery and then every 12 h.At the time of killing,blood was collected by cardiac puncture for measurement of alanine aminotransferase.The liver, kidney and spleen were then harvested for histological evaluation.2.3 RNA isolation and real-time quantitative reverse transcriptase PCR Total RNA from kidneysof ZDF rats and C57 mice was extracted using Trizol following the manufacturer's recommendation and stored at -80℃. Complementary DNA(cDNA)was reversed transcribed from the extracted RNA using First Strand Synthesis system,according to the manufacturer's instructions.Real-time reverse transcriptase PCR(RT-PCR)was performed using the TaqMan Universal PCR Master Mix and the ABI Prism 7900 Sequence Detection System.We usedβ-actin as an internal control to assure the quality of mRNAs and as a transcription level reference to measure the transcription activities of interested genes.The results were analyzed as ratios gene/β-actin and calculated threshold cycle numbers(CT)(i.e.,2-△△CT),according to the manufacturer's suggestions. 2.4 Western blot.Total protein was extracted.The lysates of kidney were stored at -80℃until assayed.A bicinchoninic acid(BCA) protein assay was used to quantify total protein in the medium according to developer's instructions.The results of the assay were measured by absorbance at 562 nm using a microplate reader.An equal amount of the whole cell protein(40μg)was denatured at 60oC for 3 min,separated by SDS-PAGE,and transferred onto nitrocellulose membranes.The blots were blocked overnight with 5%nonfat dry milk in Tris-buffered saline(TBS), followed by incubation for 1 h with the NF-Kb polyclonal antibody.The blots were washed with TBS followed by incubation with mouse anti-goat HRP-conjugated secondary antibody.Immune complexes were detected using ECL methods.The immunoreactive bands were quantified using Alphaimager 2200.Values were corrected with the absorbency of the internal control (actin).2.5 blood testing.Using high-performance liquid chromatography to measure blood glucose,cholesterol,triglyceride,serum creatinine,blood urine nitrogen,glucose tolerance test and HCT,using elisa kit to test TNF-α,IL-1,IL-6,insulin.2.6 urine testing,using elisa kit to test urine albumin and nitrate/nitrite production.2.7 blood pressure testing,using rats tail-cuff machine to test the blood pressure of rats.2.8 HE stanining:The 10%formalin-fixed,paraffin-embedded tissue sections were stained by hematoxylin and eosin.Results1.Effects on expression and activity of PPARr in mesangial cell First,hyperglycemia suppressed the pparγexpression and activity.Real- time PCR and western-blot was used to determine whether the expression of PPARγwas fluctuated in different groups.Cells cultured with high glucose expressed less PPARγcDNA by 43%(P＜0.01)and protein than that in normal glucose medium,suggesting suppression of PPARγexpression by high glucose,further,we also observed high glucose suppressed the expression of PPARγinducing gene-adipopholin gene by 70%(P＜0.01),meaning that high glucose not only downregulats the expression of PPARγbut also its activity as a transcription factor. Second,PPARγligand improved the expression and activity of PPARγin 30 mM glucose conditions.After treated with pioglitazone PPAR-γexpression was increased by 1.92(P＜0.01)when compared to that seen in the untreated cells grown in 30 mmol/L glucose,we could also observe that expression of adipophilin gene was increased by 3.74(P＜0.01) compared to the HG without treated with pioglitazone.The expression of these genes and proteins in the osmotic control groups also changed,but the level of change is less than that seen in HG with or without pioglitazone indicating that the change of genes and proteins was not due to the osmotic effects alone.2.Effects on PPARr on matrix protein production Hyperglycemia appeared to cause an increase in CⅣcollagen synthesis in the HG group compared to that seen in the NG group.HMCs showed consistently higher al CⅣmRNA expression cultured in the presence of 30 mM glucose compared to in the presence of normal glucose(1.92 P＜0.01),while it showed lower al CⅣmRNA expression when cultured in the presence of both 30 mM glucose and PIO,compared to cultures in the presence of high glucose alone(53%P＜0.01).when the cells were cultured in the presence of PIO,al CⅣprotein expression level fell signifigance than with high glucose alone. 3.Effects on PPARr ligand on matrix turnover First,effects on MMP-2/TIMP-2 signaling pathways.The cells in HG group upregulated MMP-2 mRNA expression by 1.07(P＜0.01)while PIO reduced its expression by 55%(P＜0.01)compared to cells treated with high glucose alone(Figs.3). In the case of TIMP-2,glucose alone raised mRNA expression by 2.45(P＜0.01),in association with PIO reduced the expression by 70%(P＜0.01).In order to have insights about the potential net proteolytic activity under different experimental conditions,the MMP-2/TIMP-2 mRNA balance was calculated,high glucose suppressed the protease/inhibitor balance by 40%(P＜0.01),while PIO ameliorated the balance with 29%(P＜0.05).Second,effects on MMP-9/TIMP-1 signaling pathway.HG suppressed MMP-9 mRNA expression by 39%(P＜0.01)compared to NG group while PIO and HG treated group suppressed its mRNA expression by 60%(P＜0.01)compared to without PIO group.Hyglycemia caused an augment effect on TIMP-1(0.48 P＜0.01)while PIO lead to a suppression(65%P＜0.01)compared with cells untreated with PIO,and we speculated that after treated by PIO the balance of MMP-9/TIMP-1 had been potientated by 44%(P＜0.01),this suggested that high glucose downregulated the MMP-9/TIMP-1 balance while PIO improved the balance.4.the effect of controlling diabetes progression and protecting kidney function.4.1 blood testing result,nitrooleate decreased the level of blood glucose,triglyceride and ameliorate blood glucose tolerance.4.2 urine testing result,nitrooleate decreased the urine albumin,it means that nitrooleate can protect kidney function.4.3 nitrooleate decreased the blood pressure by more than 20mmHg which means nitrooleate can important organ function,such as kidney,brain,heart,etc. 4.4 Nitrooleate can decrease body weight,nitrooleate decreased the body weight,while the rats increased body weight by more than 10g per week without nitrooleate treatment.4.5 Nitrooleate make no difference on hemoglobin,nitrooleate make no difference on hemoglobin which means it can not induce water retention.5.the effect of anti-inflammation5.1 Nitrooleate can prevention inflammation.C57 mice were pretreated by 48 hours,then injest lethal dose LPS.the mice were all alive after pretreatment with nitrooleate while the mice were all dead without nitrooleate pretreatment.5.2 Nitrooleate can protect kidney function on inflammation.C57 mice were pretreated by 48 hours,then did cecal ligation and fraction operation,after 24 hours the mice were killed and blood were selected,the blood urine nitrogen and serum critinine were tested,the blood urine nitrogen and serum critinine of treated group are significantly lower than untreated group.5.3 Nitrooleate can decrease TNF-α.Nitrooleate dramatically declines TNF-αin treated group which means nitrooleate can inhibit inflammation.5.4 Nitrooleate can decrease NF-KB expression.Nitrooleate dramatically declines TNF-αin treated group which means nitrooleate can inhibit inflammation.5.5 HE staining shows that the C57 mice without nitrooleate treatment shows dilated tubules lined with epithelial cells that show degeneration,necrosis and shedding,while the C57 mice with nitrooleate treatment do not demonstrate these histology changes.6.Nitrooleate can increase PPARα,γ,δgene expression,nitrooleate can increase PPARγexpression by about 9 fold,increase PPARαexpression by about 2 fold,increase PPARδexpression by about 2 fold.7.Nitrooleate can increase nitrogen oxide production in vivo. nitrooleate can increase nitrate/nitrite production which means it can increase nitrogen oxide production in vivo.Conclusions1.it is tempting to postulate that TZD have pharmacological effect on mesangial cell cultured in high glucose.They inhibit synthesis of collagen typeⅣ;TZD ameliorate the balance of MMP-2/TIMP-2 and MMP-9/TIMP-1. In view of our new findings and in consideration that pioglitazone is already orally administered,this drug which have been used in over two million people with minimal side effects hold great promise to complement conventional chemotherapeutic regimens for diabetic nephrology therapy.2.we speculate nittooleate may be a therapeutic compound for diabetic nephropathy and it has definite anti-inflammation function.