The Mechanism Of Peroxynitrite Mediating Kidney Lesion Of Diabetes

Objectives: Diabetes mellitus (DM) is one of the three serious diseases having the highest case-fatality rate, diabetic patient have the three times risk of blood vessel complication (including microvascular pathological changes and macrovascular pathological changes) than non-diabetic patient. In diabetic patient, diabetic nephropathy (DN) is one of the common and obstinate microvascular chronic complications, which is also the main cause leading to death and remnant for diabetes. Many factors are involved in the pathogenesis of this disease, such as heredity background, disturbance of carbohydrate metabolismn or lipid metabolism, and blood dynamic abnormality. Among them, oxidative stress has been considered to be one of the important mechanism involved in DN. Oxidative stress usually refers to a situation due to a serious imbalance between the production of free radicals and anti-oxidant defense mechanisms or excessive oxidant received in vivo, which cause more production of reactive oxygen species (ROS) or reactive nitrogen species (RNS), then finally lead to cytotoxicity. Peroxynitrite (ONOO-) is one of the RNS important members. It has been reported that in diabetes, enhanced generation of nitric oxide caused by inducible nitric oxide synthase (iNOS), can rapidly react with free radicals superoxide to form peroxynitrite. Peroxynitrite can oxidize and/or nitrate many biomolecules, which will lead them to reconstruction or dysfunction. For example, tyrosine residue of protein can be nitrated by peroxynitrite to form 3-nitrotyrosine (3-NT). Thus the formation of 3-nitrotyrosine is suggested as a marker for ONOO-. At present, ONOO- has been indicated to be involved in many diseases, such as atherosclerosis, acute lung injury, septicemia, shock, ischemia/reperfusion injury and Parkinson's disease, especially diabetes and its complications. However, the contributions of peroxynitrite to DN and the underlying mechanism have not been fully explored.Pathological roles of peroxynitrite include: inhibition of protein activity, leading to lipid peroxidation, DNA injury and mitochondrial dysfunction, inducing cell apoptosis or death. Recent studies have show that peroxynitrite also play an important role in signal-transduction pathways, including mitogen-activated protein (MAP) kinase, PI3K/Akt, and NFκB etc. Peroxynitrite was involved in many diseases by activating or inhibiting signal-transduction. Recently, Platt DH etal found that peroxynitrite can increase vascular endothelial growth factor (VEGF) expression in vascular endothelial cells via activating STAT3. Notably, activation of the Janus kinase (JAK) /? signal transducer and activators of transcription (STAT) signaling cascade results in excessive proliferation and growth of glomerular mesangial cells (MC), more production of transforming growth factor1 (TGF-β1) and fibronectin (FN), which may contribute to DN. Whether the pathogenetic role of peroxynitrite in DN also be via JAK/STAT pathway remains unclear.It has been reported that activating of TGF-β1 by JAK/STAT pathway play an important role in the pathogenesis of DN. TGF-β1 can stimulate mesangial cells to synthesize extracellular matrix protein (ECM) such as fibronectin, then lead to basement membrane thickened, mesangial matrix increased and fibrosis. Thus one of the basic underlying mechanisms of diabetic nephropathy seems involved in more production of TGF-β1 and extracellular matrix molecules such as fibronectin. Moreover, TGF-β1 also mediate many MC basic biological behaviour in high glucose environment, such as proliferation, hypertrophy and apoptosis. For example, TGF-β1 can induces expression of cyclin-dependent kinase inhibitors (CKI). The CKI can bind to cyclin/CDK complexes, inhibit their activity, thereby prevent G1/S-phase transit and inhibit cell proliferation. Meanwhile, Khera T etal found that TGF-β1 can lead MC to apoptosis. It has been demonstrated that MC basic biological behaviour play an important role in DN. Since great significant effect of TGF-β1 in stimulating ECM and mediating MC basic biological behaviour, Whether the pathogenetic role of peroxynitrite in DN be via inducing TGF-β1 after stimulating JAK/STAT pathway remains unexplored.In this study, Wistar rats were used to induce experimental diabetes model, human glomerular mesangial cells (HMC) were cultured in high glucose (HG) in vitro. The expression of ONOO-, JAK/STAT pathway, TGF-β1 and FN in rats renal cortex and HMC induced by HG were systemic studied at different levels from whole, cells and molecules. The affection of HG to HMC's proliferation and apoptosis were also investigated. Meanwhile, Uric acid (UA, a specific ONOO- scavenger) and AG490 (a specific JAK2 inhibitor) were used to measure the change of accordingly target. So as to discuss the role of ONOO- in the pathogenesis of DN and whether its role in DN be via stimulating JAK/STAT pathway and inducing TGF-β1, FN. It is expected to provide a new thinking for the role of ONOO- in the DN, its molecule mechanism and the prevention and cure of DN.Methods:1 Animal and blood/urine biochemical markerWistar male rats were divided randomly into 3 groups: Control group, Diabetes group (DM) and Urate group. DM group were injected intraperitoneally with STZ (40mg/kg, dissolved in 0.1mol/L citrate buffer, pH4.4) to induce diabetes. Control group were injected with equivalent citrate buffer. Rats in Urate group were injected intraperitoneally with STZ (40mg/kg) and fed urate solution (160mg/kg/day), rats in Control group and DM group fed with equivalent water. 3 days after injection, diabetes was confirmed by the concentration of fasting blood-glucose higher than 13mmol/L and urine glucose (+++) or above. Rats in the 4 groups were all fed normal diet. After raised for 13 weeks, 24h urine of five rats from each group was respectively collected by metabolic cages for the detection of urine protein (Upro) and urine creatinine (Ucr). Blood was collected from femoral artery, serum was separated for the detection of blood glucose (Glu), blood urea nitrogen (BUN) and serum creatinin (Scr).2 Detection of routine morphology change and the expression of ONOO-, JAK/STAT pathway, TGF-β1 and FN in rat renal cortexFive rats from each group (Control group, DM group and Urate group) were respectively weighted and sacrificed after raised for 13 weeks. Partial renal tissures were fixed in 4% formaldehyde and 2% glutaraldehyde for light microscopic, electron microscopic observation and immunohistochemical staining for the detection of NT total protein (a marker for ONOO-) expression. Partial renal cortices were used to abstract total protein for the detection of NT total protein, JAK2, p-JAK2, STAT1, p-STAT1, STAT3, p-STAT3, TGF-β1 and FN by western blot. Partial renal cortices were used to abstract total RNA for the detection of TGF-β1 mRNA and FN mRNA by reverse transcription and polymerase chain reaction (RT-PCR).3 Expression of ONOO-, p-JAK2/p-STAT3, TGF-β1 and FN protein in HMCHuman glomerular mesangial cells (HMC) were incubated with RPMI 1640 contianing 10% fetal bovine serum, 100kU/L penicillin and 100mg/L streptomycin. Before stimulation, HMCs were incubated with serum-free RPMI 1640 for 24 hours to synchronize the cell growth. Then, the HMC were divided into five groups: NG group (5.5mmol/L glucose); HG group (30mmol/L glucose); Mannitol hyperosmotic control group (5.5mmol/L glucose+24.5mmol/L mannitol); HG+AG490 group (30mmol/L glucose+10μmol/L AG490); HG+UA group (30mmol/L glucose+100μmol/L uric acid). The cell morphology changes were observed by inverted microscope at 12, 24, 48 hours after incubation. Cells were harvested to abstract total protein at 12, 24 and 48 hours after incubation. The medium was collected for detecting the protein level of TGF-β1 and FN. The expression of NT total protein, p-JAK2, p-STAT3, TGF-β1proteins were examined by immunocytochemistry and NT total protein, p-JAK2, p-STAT3 proteins by Western-blot.4 Proliferation, apoptosis of HMCHMCs were cultured in 96-pore plate, with each pore having 200μL volumn and 4000/well density. After 24h of cells plating, medium was sucked out, HMC were then incubated with serum-free RPMI 1640 for 24 hours to synchronize the cell growth. After that, cells were divided into four groups: NG group, HG group, Mannitol hyperosmotic control group and HG+UA group. MTT assay was used to investigate the proliferation of HMCs in different groups at different time (12h, 24h and 48h). When density of HMCs in 25cm2 culture flask reached to 70-80%, HMC were incubated with serum-free RPMI 1640 for 24 hours, then divided into five groups (NG group, HG group, Mannitol hyperosmotic control group, HG+AG490 group and HG+UA group). Flow cytometry (FCM) was used to investigate cell apoptosis and cell cycle in different groups at 48h.Results:1 The expression of ONOO-, JAK/STAT pathway, TGF-β1 and FN in diabetic rat renal cortex①Pathological morphological changes: The pathological changes evaluated by HE and PAS staining showed that there were no pathological changes in control group, however, in DM group, mesangial matrix was increased, some capillaries of glomerulu were enlarged, glomerular and renal tubular basement membrane was thickened, glomerular sclerosis can also be observed in some glomerulus. The pathological changes evaluated by TEM showed that compared with control group, glomerular ultrastructure in DM group was disordered with mesangial matrix increased, basement membrane thickened and podocyte foot-process fused mostly.②Characterization of renal function: Compared with control group, in DM group creatinine clearance rate was decreased, the level of blood glucose, blood urea nitrogen and 24h urine protein were increased significantly.③Immunohistochemical analysis: In control group, staining of NT total protein was weak-positive. Compared with control group, in DM group the expression of NT total protein increased, positive staining (brownish-yellow) of NT total protein was observed mainly in cytoplasim and nucleus of glomerular cells.④Western blot analysis: Compared with control group, in DM group the expression of NT total protein, p-JAK2, p-STAT1, p-STAT3, TGF-β1 and FN were increased respectively. There was no obvious difference of JAK2, STAT1 and STAT3 content between control group and DM group.⑤RT-PCR analysis: TGF-β1 mRNA and FN mRNA expressed higher in DM group than control group.2 The effect of UA on the expression of ONOO-, JAK/STAT pathway, TGF-β1 and FN in diabetic rat renal cortex①Pathological morphological changes: Treatment of UA significantly attenuated the glomerular pathological change in DM group, that is, mesangial matrix was decreased, basement membrane was attenuated and the degree of foot-process fusion was alleviated.②Characterization of renal function: Compared with DM group, in UA group creatinine clearance rate was increased, blood urea nitrogen and 24h urine protein were decreased.③Immunohistochemical and Western blot analysis: UA could significantly decreased the high expression of NT total protein, p-JAK2, p-STAT1, p-STAT3, TGF-β1 and FN in DM group.④RT-PCR analysis: TGF-β1 mRNA and FN mRNA expressed lower in UA group than DM group.3 The expression of ONOO-, JAK/STAT pathway, TGF-β1 and FN induced by HG in HMC and the effect of HG on the proliferation/apoptosis of HMC①HMC morphological changes: Under inverted microscope, the HMCs stimulated by HG grew longer and revealed the decreased or shortened cell process, appeared many granula and vacuole in cytoplasm.②Immunocytochemical staining: In HG group, the expression of NT total protein, p-JAK2, p-STAT3, TGF-β1 protein increased significantly compared with control group (negtive or weak expression). The positive granula of NT total protein, p-JAK2 and p-STAT3 were mainly in cytoplasm, partly in nucleus, TGF-β1 mainly in cytoplasm.③Western blot analysis: The expression of NT total protein, p-JAK2 and p-STAT3 protein in HG group were higher than control group (weak expression). In HG group, the expression of protein (same as above) increased with time, and reached to highest level at 48h after stimulation.④ELISA assay: In HG group, the content of TGF-β1 and FN increased with time (12h, 24h and 48h), and were significantly higher than control group at 12h, 24h or 48h respectively after stimulation.⑤MTT assay: Compared with control group (OD:0.636±0.047), the proliferation of HMC at 48h after stimulation was significantly inhibited in HG group (OD:0.362±0.022), there was no obvious difference of proliferation between control group and HG group at 12h or 24h respectively.⑥FCM assay: Cell apoptoic rate was much higher in HG group at 48h (11.53±2.16 %) than that of control group (1.23±0.07 %). The percentage of HMC in G0/G1 phase increased at 48h after stimulation, meanwhile HMC in S phase and G2/M phases decreased significantly.4 The effect of UA on the expression of ONOO-, JAK/STAT pathway, TGF-β1 and FN induced by HG in HMC and the effect of UA on the proliferation/apoptosis of HMC①HMC morphological changes: In HG+UA group, the HMCs had the similiar morphology with control group. Compared with HG group, cells grew shorter, revealed more cell process and appeared less granula in cytoplasm.②Immunocytochemical staining: In HG+UA group, the expression of NT total protein, p-JAK2, p-STAT3, TGF-β1 protein decreased significantly compared with HG group.③Western blot analysis: The expression of NT total protein, p-JAK2, p-STAT3 protein in HG+UA group were lower than HG group at the corresponding time.④ELISA assay: UA could effectively down-regulate the content of TGF-β1 and FN in HG group at 12h, 24h or 48h respectively.⑤MTT assay: Compared with HG group (OD:0.362±0.022), the proliferation of HMC at 48h after stimulation increased in HG+UA group (OD:0.698±0.014).⑥FCM assay: In HG+UA group, cell apoptosis was suppressed (4.61±0.39 %) than HG group (11.53±2.16 %) at 48h, the percentage of HMC in G0/G1 phase decreased, meanwhile HMC in S phase and G2/M phases increased.5 The effect of AG490 on the expression of ONOO-, JAK/STAT pathway, TGF-β1 and FN induced by HG in HMC①Immunocytochemical staining: In HG+AG490 group, the positive expression of p-JAK2, p-STAT3, TGF-β1 protein decreased significantly compared with HG group.②Western blot analysis: The expression of p-JAK2, p-STAT3 protein in HG+AG490 group were lower than HG group at the corresponding time. However, there was no obvious difference of NT total protein expression between HG+AG490 group and HG group.③ELISA assay: AG490 could effectively down-regulate the content of TGF-β1 and FN in HG group at 12h, 24h or 48h respectively.④FCM assay: In HG+AG490 group, cell apoptosis was suppressed (5.70±0.72 %) than HG group (11.53±2.16 %) at 48 hour.Conclusions:From above results we can draw the following conclusions:1 The overexpression of NT total protein(the marker of ONOO-)may be related to the progression of diabetic nephropath (DN), the application of UA (a specific ONOO- scavenger) may be play an important role in prevention and cure of DN.2 JAK/STAT pathway may be closely involved in the progression of DN, scavenging of ONOO- may be related to the inhibition of JAK/STAT pathway, ONOO- may be involved in the progression of DN via activaing JAK/STAT pathway.3 The overexpression of TGF-β1 and FN may be one of the mechanism involved in DN, overexpression of TGF-β1 and FN may be related to the activation of JAK/STAT pathway, exaggerated ONOO- may mediate the lesion of DN via overexpression of TGF-β1 and FN. 4 ONOO- may play an important role in HMC basic biological behaviour (proliferation, apoptosis and cell cycle) in high glucose environment.