| Objectives:In development countries,diabetes mellitus(DM) is the leading cause of chronic renal failure and is increasing as a cause of morbility and mortality worldwide. Both type 1 and 2 diabetes play important role in the development of its microvascular complications. Among all these complications, diabetic nephropathy(DN) has become the principal cause of end-stage renal failure and cardiovascular mortality. Many mechanisms were induced in pathogenesis and progression of DN, included changes of metabolism and haemodynamics, inflammation and oxidative stress and activation of the renin angiotensin system. The role of inflammation and oxidative stress is the most important.The reactive oxygen species(ROS) is the main driving force in a cascade of damaging effects mediated by high glucose. High glucose in renal proximal tubular cells stimulates the production of advanced glycation end products(AGEs), the angiotensin II(Ang II) and activation of PKCs, all of which lead to increased production of free radicals caused oxidative stress. Meanwhile,accumulating evidence now indicates that inflammatory mechanisms,proinflammatory cytokines and profibrotic growth factors have all been implicated in the high glucose-induced renal dysfunction. Prolonged hyperglycemia exerts an important diversity of actions mediated by cytokines and growth factors that produces oxidative stress and chronic inflammatory elements from the initial tubular functional abnormalities to late stages of tubulointerstitium. Oxidative stress is another pathway that leads to inflammation through activation of JNK, NF-κB, and p38 mitogen-activated protein kinase(p38MAPK) lead to kidney injury. But the mechanism of inflammation and oxidative stress is still unclear.CD36, a multiligand class B scavenger receptor, was originally describedas a platelet receptor glycoprotein IV has been shown to be expressed by multiple cell types, such as macrophages, microvascular endothelial cells,platelets and epithelial cells. Previous studies have demonstrated that CD36 participates in multiple biological functions. In particular, CD36 may be a key mediator in mediating the production of inflammatory and ROS in chronic kidney disease(CKD), stroke, atherosclerosis and obesity. In human diabetic nephropathy, CD36 is involved in the mechanisms of apoptosis. CD36 is also a potential anti-fibrogenic target in albumin-Induced renal proximal tubule fibrosis. CD36 is present on renal tubular epithelial cells under high glucose condition, meanwhile, high glucose condition characterized by inflammation,and oxidant stress. These raise the possibility that CD36 may be a potential modulator of inflammatory and oxidative stress in high glucose-induces renal tubular epithelial cells injury.SS-31 peptide(D-Arg-2’, 6’-dimethyltyrosine-Lys-Phe-NH2) belongs to a family of small cell-permeable peptides that target and concentrate in the inner mitochondria membrane, the site of ROS generation. This peptide can scavenge ROS and reduce mitochondrial ROS production and thereby prevent mitochondrial permeability transition. SS-31 has been shown to readily penetrate intact islets, prevent mitochondrial depolarization, enhance islet cell yield, reduce apoptosis, and improve posttransplantation function. SS-31 has been shown to reduce the cell death and production of ROS in mitochondria,stabilized mitochondrial potential in HG-treated human retinal endothelial cells. Previous studies showed that SS-31 inhibited renal tubular apoptosis,oxidative stress, inflammatory response and damage in ischemia-reperfusion injury and unilateral obstruction rats. Our previous studies also showed that SS-31 inhibited renal tubular production of ROS, prevent mitochondrial permeability transition and inhibited renal tubular apoptosis. New studies showed that SS-31 attenuates ischemic brain injury by down-regulating CD36.So, we speculate that the role of SS-31 in diabetic renal damage maybe act as the same way.In this study we intend to further observation, based on the original workof the role of CD36 in diabetic renal tubular injury, and to investigate the incidence of diabetic nephropathy, the development of mechanisms of action and to find a new target for the treatment of diabetic nephropathy. In addition,we also investigate the relationship of SS-31 and CD36, in order to provide a scientific basis for targeting drugs.Methods:1 Determination of CD36 in patients with diabetic nephropathy, db/db mouse and HK-2 cells.Ten patients diagnosed as developed diabetic nephropathy by renal biopsy and clinical data in this study. The renal tissues(n=10) obtained from distant portions of kidneys surgically excised because of the presence of a localized neoplasm were used as control(>5cm). Protein expression of CD36 was assessed by immunhistochemical staining.Male db/db diabetic mice were 8 week-old. The same week-old male db/m mice are used as normal controls. The mice of db/m group and db/db group were feed normally(room temperature 22±2°C, humidity 55±2%). Mice were sacrificed at 20 week-old. Blood and urine samples were collected.Partial renal tissures were fixed in 4% neutral formalin for histochemical.Protein and RNA were extracted from partial renal cortices for Western blot and Real-time PCR.The HK-2 cells were cultured in DMEM medium supplemented with 5%fetal bovine serum, 2 m M L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin in a 95% air, 5% CO2 atmosphere. HK-2 cells were randomly divided into 2 groups: normal glucose group(5.5 mmol/L glucose, NG), high glucose group(30 mmol/L glucose, HG), The groups were cultured for 0, 6,12,24, 48 and 72 hours respectively, and then HK-2 cells were harvested.2 Knockdown of CD36 impact on high glucose-induced inflammatory and oxidative stress in HK-2 cellsTansfections of HK-2 cells with CD36 si RNA plasmid according to the manufacturer’s instructions. The HK-2 cells were cultured in DMEM medium supplemented with 5% fetal bovine serum, 2 m M L-glutamine, 100 U/mlpenicillin and 100 μg/ml streptomycin in a 95% air, 5% CO2 atmosphere.HK-2 cells were randomly divided into 5 group: normal glucose group(5.5mmol/L glucose, NG), normal glucose+mannitol group(5.5 mmol/L glucose+24.5 mmol/L mannitol, M), high glucose group(30 mmol/L glucose,HG), high glucose+CD36 si RNA(30 mmol/L glucose+ CD36 si RNA, si RNA),high glucose+SSO(30 mmol/L glucose+0.5m M SSO, HG+SSO), The groups were cultured for 48 hours, and then HK-2 cells were harvested. The protein expression of CD36、NF-k B、histone3、p-iκB、Total iκB、ERK1/2、p-ERK1/2、Collagen I、 Fibronectin、α-SMA、E-cadherin、TGF-β1、CTGF,Smad2 and p-Smad2 was detected by Western blot. The m RNA levels of CD36、α-SMA、E-cadherin 、 TGF-β1and CTGF were evaluated by Real-time PCR. The secretion of IL-1β、MCP-1、TNF-α、Collagen I、 Fibronectin in HK-2 cells were examined by ELISA. The expression of CD36、NF-k B、α-SMA and E-cadherin in HK-2 cells were examined by immunofluorescence staining.The levels of ROS in HK-2 cells were evaluated by flow cytometry.In order to investigate the relationship between CD36 and oxidative stress, we use NAC and Tempo to inhibit the production of ROS. HK-2 cells were randomly divided into 6 group: normal glucose group(5.5 mmol/L glucose, NG), normal glucose+mannitol group(5.5 mmol/L glucose+24.5mmol/L mannitol, M), high glucose group(30 mmol/L glucose, HG), high glucose+NAC(30 mmol/L glucose+ 5 mmol/L N-Acety-L –Gysteine,NAC),high glucose+Tempol(30 mmol/L glucose+2 mmol/L Tempol,Tempol), high glucose group(30 mmol/L glucose, HG), high glucose+NAC +Tempol(30mmol/L glucose+ 5 mmol/L N-Acety-L –Gysteine+2 mmol/L Tempol, NT),The groups were cultured for 48 hours, and then HK-2 cells were harvested.The protein expression and m RNA levels of CD36 were examined by western blot and Real-time PCR.3 Role of SS-31 in db/db mice and high glucose induced HK-2 cells.Male db/db diabetic mice were randomly divided into two groups:diabetic group(db/db group) and diabetic+SS-31 group(db/db+SS-31). The same week-old male db/m mice as normal controls(db/m) and SS-31 group(db/m+ SS-31 group, db/m+ SS-31). The mice of db/db+ SS-31 group and db/m+ SS-31 group were administered daily with SS-31(3 mg/kg) by intraperitoneal injection. The mice of db/m group and db/db group were only administered daily with the same volume of normal sodium by SS-31. Mice were sacrificed at 20 week-old. Blood and urine samples were collected for examination of Glu, UPE and Scr. PAS stains were used for morphological changes. The expression of Mn SOD、CAT、NOX4、p22、CD36 and NF-κB protein was respectively evaluated by Western blot. The m RNA levels of Mn SOD、CAT、NOX4、p22 and CD36 were evaluated by Real-time PCR. The expression of CD36 was evaluated by immunohistochemistry.HK-2 cells were randomly divided into 4 group: normal glucose group(5.5 mmol/L glucose, NG), normal glucose+mannitol group(5.5 mmol/L glucose+24.5 mmol/L mannitol, M), high glucose group(30 mmol/L glucose,HG), high glucose+SS-31(30 mmol/L glucose+ 100 n M SS-31, SS-31). The groups were cultured for 48 hours, and then HK-2 cells were harvested. The protein expression of Mn SOD、CAT、NOX4、p22、CD36 and NF-k B was detected by Western blot. The m RNA levels of Mn SOD、CAT、NOX4、p22and CD36 were evaluated by Real-time PCR. The expression of CD36 and NF-k B were evaluated by immunofluorescence staining. The levels of ROS in HK-2 cells were evaluated by flow cytometry.Results:1 The expression of CD36 in diabetic nephropathy1 There were no abnormal changes in glomerulus, renal tubule and interstitium of control group by light microscopy. Pathological changes including glomerular enlargement, increase of glomerular basement membrane in thickeness, increase of ECM, the presence of KimmelstielWilson lesions, focal tubular epithelial vacuolar degeneration as well as interstitial fibrosis were observed in the patients of diabetic nephropathy. 2In control group, the levels of of CD36 expression were low. Compared with control group, the expression of CD36 was increased significantly(P<0.05).The expression of CD36 was in renal tubular epithelial cell. 3Compared withcontrol group, the FBG, weight levels, 24 h urine protein, BUN and Scr was significantly increased in db/db group than db/m group(P<0.05).4Compared with control group, the expression of CD36 was increased significantly in db/db group than db/m group(P<0.05). The expression of CD36 was in renal tubular epithelial cell. 5The levels of CD36 protein and m RNA were increased significantly in after cultured for 48 hours in high glucose. The expression of CD36 was in cytomembrane.2 Knockdown of CD36 ameliorates high glucose-induced inflammatory and oxidative stress in HK-2 cells1Compared with control group, the expression of IL-1β、MCP-1、TNF-αwere increased significantly in high glucose-induced HK-2 cells, and inhibited by transfection of CD36 si RNA plasmid or SSO treatment(P<0.05).2Compared with control group, the actvation of NF-κB 、iκB and ERK1/2were increased significantly in high glucose-induced HK-2 cells, and inhibited by transfection of CD36 si RNA plasmid or SSO treatment(P<0.05).3Compared with control group, the levels of ROS were increased significantly in high glucose-induced HK-2 cells, and inhibited by transfection of CD36 si RNA plasmid or SSO treatment(P<0.05).4Compared with control group, the expression of CD36 was increased significantly in high glucose-induced HK-2 cells, and inhibited by NAC and/or Tempo treatment(P<0.05).3 Knockdown of CD36 ameliorates high glucose-induced epithelial-mesenchymal transition in HK-2 cells1The morphology of HK-2 cells in NG group was normal. HK-2 cells of HG group were long and thin.2Compared with control group, the expression of Collagen I and Fibronectin were increased significantly in high glucose-induced HK-2 cells, and inhibited by transfection of CD36 si RNA plasmid or SSO treatment(P<0.05). 3Compared with control group, the expression of α-SMA was increased and the expression of E-cadherin was decreased significantly in high glucose-induced HK-2 cells, and reversed by transfection of CD36 si RNA plasmid or SSO treatment(P<0.05). 4Comparedwith control group, the levels of TGF-β1 and CTGF were increased significantly in high glucose-induced HK-2 cells, and inhibited by transfection of CD36 si RNA plasmid or SSO treatment(P<0.05). 5Compared with control group, the phosphorylation of Smad2 was increased significantly in high glucose-induced HK-2 cells, and inhibited by transfection of CD36 si RNA plasmid or SSO treatment(P<0.05).4 Role of SS-31 in db/db mice and high glucose induced HK-2 cells.1Diabetic db/db mice showed slightly glomeruli hypertrophy, increasing mesangium matrix, thickened glomerular basement membrane, reduction in the number of renal tubular epithelial cell, partial tubular epithalial vacuolar degeneration by light microscope. Compared with db/m group, 24 h urine protein, BUN and Scr was significantly increased in db/db group and was significantly decreased in SS-31 group. 2 Compared with db/m group, the levels of MAD and 8-OHd G were increased significantly in db/db group.Treatment with SS-31 induced a significant decrease of MDA and 8-OHd G levels compared to the db/db group. 3 Compared with db/m group, the expression of Mn SOD and CAT were decreased and the expression of NOX4,p22, CD36 and NF-κB were increased significantly in db/db group, and reversed by SS-31 treatment. 4Compared with NG group, the levels of ROS were increased significantly in high glucose-induced HK-2 cells, and inhibited by SS-31 treatment(P<0.05).5Compared with NG group, The expression of Mn SOD and CAT were decreased and the expression of NOX4, p22, CD36 and NF-κB were increased significantly in high glucose-induced HK-2 cells,and reversed by SS-31 treatment(P<0.05).Conclusions:1 The overexpression of CD36 is found in renel tissues from patients with diabetic nephropathy, diabetic mice and high glucose induced HK-2 cells by which suggests that CD36 may mediate renal tubular injury of diabetic nephropathy.2 Knockdown of CD36 prevents high glucose-induced secretion of inflammatory factor, inhibiting activation of inflammation related signalingpathway. These results suggest that the overexpression of CD36 is related with inflammatory in HK-2 cells.3 Knockdown of CD36 prevents high glucose-induced production of ROS. The overexpression of CD36 induced by high glucose can be inhibited by NAC and Tempo. These results suggest that CD36 expression is ROS-dependent, knockdown of CD36 prevents high glucose-induced production of ROS.4 Knockdown of CD36 prevents high glucose-induced expression and secretion of Collagen I and fibronectin, inhibiting EMT and activation of fibrosis related signaling pathway. These results suggest that the overexpression of CD36 is related with fibrosis in HK-2 cells.5 SS-31 treatments significantly alleviated renal hypertrophy, UAE and creatinine in db/db mice. SS-31 also inhibited oxidative stress, NADPH oxidase activation, expression of CD36 and NF-κB p65 and promoted the activity of Mn SOD and CAT in db/db mice and HG-induced HK-2 cells.These findings suggest that SS-31 provides a treatment for diabetic nephropathy and through a CD36-dependent pathyway. |
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