| Background:Chronic kidney disease (CKD) is a world-wide health problem and the prevalence of patients with CKD is increasing rapidly. Progressive renal diseases are characterized by an interstitial infiltrate of mononuclear cells and the tubulointerstitial fibrosis. It has been found that there are positive correlations between the degree of tubulointerstitial fibrosis and the decline of renal function. The perturbation of tubular epithelial cells plays a key role in the development of tubulointerstitial fibrosis. A growing body of evidence indicates that activation of the renin angiotensin system (RAS) and the subsequent generation of angiotensinⅡ(AngⅡ) may directly or indirectly be responsible for renal proximal tubular cell perturbation and the tubulofibrosis in CKD. For example, in vitro studies have shown that murine proximal tubular cells cultured in the presence of AngⅡexhibit increased expression of TGF-beta 1 and inflammation factors thus induced the tubular perturbation. Moreover the clinical observation demonstrated that angiotensin-converting enzyme (ACE) inhibition reduces proteinuria and limits progressive deterioration of renal function in a great variety of renal diseases, independently of the presence of hypertension.The presence of a local intrarenal RAS has been generally accepted. The messenger RNA (mRNA) components of the RAS, including angiotensinogen (AGT), renin, ACE, and AngⅡreceptors (AT-1 and AT-2 receptors), are expressed in murine, mouse, and rat immortalized proximal tubular cell lines. Previously studies indicated that the AGT protein is secreted from rat immortalized renal proximal tubular cells; supporting the idea that intrarenal AngⅡis derived from the AGT that is synthesized within the renal proximal tubular cells in vivo. Enhanced local formation of AngⅡmay therefore contribute to the pathogenesis of CKD.CKD is associated with increased modification of proteins. A typical representation is the formation and accumulation of advanced glycation end products (AGEs), the non-enzymatic glycation protein products induce tubular perturbation mainly through interaction of AGEs with the cell surface receptor for AGEs (RAGE). In addition to AGEs, a family of oxidized protein compounds, termed advanced oxidation protein products (AOPPs), has emerged as a novel class of renal pathogenic mediators. AOPPs are the dityrosine-containing and cross-linking protein products formed during oxidative stress by reaction of plasma protein with chlorinated oxidants. Plasma AOPPs are mainly carried by albumin, their concentration closely correlating with the level of dityrosine, a hallmark of oxidized protein. Therefore, AOPPs have been considered as the markers of oxidant-mediated protein damage.Accumulation of AOPPs has been found in patients with diabetes, and nondiabetic kidney diseases such as IgA nephropathy, in whom the level of AOPPs was a strong predictor for progression of renal disease. Our recent in vivo studies demonstrate that elevation of plasma AOPPs is associated with interstitial infiltrate of mononuclear cells and the tubulointerstitial fibrosis in streptozotocin-induced diabetic rats and nondiabetic remnant kidney model. And our in vitro experiments revealed AOPP activate vascular endothelial cells via a RAGE-mediated, NAD (P) H oxidase-dependent signaling. Yasunori Iwao etc proved that AOPPs induced the injury of renal tubular cell through CD36. Although emerging evidence recognized AOPPs as renal pathogenic factors and supported a role for AOPPs in mediating tubular cell perturbation, the underlying mechanism remains unclear. It is still unknown whether AOPPs can activate RAS in tubular cell therefore inducing tubular cells perturbation, if it is the case, what about its intracellular signal transduction processes. In this study, we focused on elucidating the activation of RAS and the signaling pathway activated by AOPPs-albumin in rat proximal tubular cells. Our data indicate that AOPPs, either in vitro or in vivo, induced the activation of RAS likely through a CD36, RAGE-mediated signals involving PKC alpha, NADPH oxidase dependent ROS generation, activator protein-1(AP-1) and nuclear transcription factorκB (NF-κB).Methods1. Preparation of AOPPAOPP was prepared in vitro according to the method described by Witko-Sarsat and our laboratory. AOPPs content was determined by measuring absorbance at 340 nm in acidic condition and was calibrated with Chloramines-T in the presence of potassium iodine.2. Cell experiment1) cell cultrueA rat kidney tubular epithelial cell line (NRK52E) was obtained from ATCC (Manassas, VA, USA) and maintained in DMEM/F-12 containing 10% Fetal Bovine Serum (Gibco BRL, Grand Island, NY, USA), 100U/ml penicillin and 100μg/ml streptomycin. Passages6-10 cells were used for the experiments. For all experiments, the cells were grown to 80%-90% confluence on 6-well plates or 100×20mm plastic petri dishes (Nunc, Roskilde, Denmark) and maken quiescent by incubation in serum-free DMEM for 24 hours before stimulation with AOPP or RSA. All reagents used were certified to be endotoxin free.2) AOPP-RSA induced the activation of RAS kidney tubular epithelial cell. In brief, cells were treated with AOPP in concentrations ranging from 50 to 200μg/ml and incubated for various time periods up to 24 hours, and then the mRNA levels of AGT, ACE, AT1 were analyzed by RT-PCR; the protein levels of AGT, ACE, AT1 were analyzed by western blot; the angiotensinⅡconcentrations in cell lysate and medium culture were determined by Elisa; the ACE activity in cell lysate and medium culture were measured by a fluospetrometer. 3) AOPP activated PKC in renal tubular epithelial cell. In brief, cells were incubated with AOPP for various time periods up to 30min, and then the activation of PKC isoforms were analyzed by western blot.4) AOPP induced the production of ROS in renal tubular epithelial cell. In brief, cells were treated with AOPP in concentrations ranging from 50 to 200μg/ml and incubated for various time periods up to 1 hour, and then intracellular ROS production was assessed by fluorescence of DCF. To verify NAD(P)H oxidase contribution, cells were pre-treated with c-SOD(200U/ml), diphenylene iodonium (DPI,10μmol),apocynin(100μmol), rotenone(2μmol),TTFA(10μmol),myxothiazol(10μM),L-NAME(inhibitor of nitric oxide synthase, 100μmol), oxipurinol(100μmol), indomethacin (10μM),and for one hour at 37℃respectively before challenged by AOPP, then intracellular ROS production was measured.5) AOPP activated NADPH oxidase in renal tubular epithelial cell. In brief, cells were incubated with AOPP for various time periods up to 60min, then phosphorylation of p47phox, binding of p22phox to p47phox, Nox4 or Nox2, and experession of these proteins were detected by immunoprecipitation and western blot.6) AOPP activated AP-1 in renal tubular epithelial cell. In brief, cells were incubated with AOPP for various time periods up to 60min, then phosphorylation of c-jun, and experession of c-jun and c-fos were detected by western blot.7) AOPP activated NF-kB in renal tubular epithelial cell. In brief, cells were incubated with AOPP for various time periods up to 60min, and then phosphorylation of p65 was detected by western blot.8) AOPP activated RAS through CD36 and RAGE in renal tubular epithelial cell. Western blot analysis indicated that megalin, CD36, SR-BI, Lox-1, galection-3, RAGE and SR-A were expressed in renal tubular epithelial cell. To veriy the role of CD36 and RAGE, cells were transfected with siRNAs of megalin, CD36, RAGE, SR-BI, Lox-1, galection-3, and SR-A respectively, or preincubated with anti-CD36 or anti-RAGE antibody before AOPP stimulation, then western blot was used to detect the protein levels of AGT and AT1.9) AOPP activated PKC isoforms through CD36 and RAGE in renal tubular epithelial cell. Renal tubular epithelial cells or renal tubular epithelial cells transfected with CD36 siRNA were exposed to 100μg/ml AOPP for 30min in the presence or absence of anti-RAGE antibody, then western blot was employed to detect the membrane phosphorylation of PKC alpha.10) AOPP activated NADPH oxidase through CD36 and RAGE in renal tubular epithelial cell. Normal cells or cells transfected with CD36 siRNA were exposed to 100μg/ml AOPP for 30min in the presence or absence of anti-RAGE antibody, then western blot was employed to detect the phosphorylation of p47phox.11) AOPP induced the production of ROS through CD36 and RAGE in renal tubular epithelial cell. Normal cells or cells transfected with CD36 siRNA were exposed to 100μg/ml AOPP for 30min in the presence or absence of anti-RAGE antibody, then intracellular ROS production was assessed by fluorescence of DCF.12) The relationship between PKC and NADPH/ROS. AOPP induced the activation of NADPH oxidase and production of ROS through PKC alpha, and blockade of NADPH oxidase and ROS generation partly inhibited the activation of PKC alpha. Renal tubular epithelial cells were exposed to 100μg/ml AOPP for 30min in the presence or absence of Go6983, Go6976, then activation of NADPH oxidase was detected by immunoprecipitation and western blot; and intracellular ROS production was assessed by fluorescence of DCF. Cells were exposed to 100μg/ml AOPP for 30min in the presence or absence of c-SOD, DPI and apocynin, and then western blot was employed to detect the membrane phosphorylation of PKC alpha.13) AOPP activated AP-1 and NF-kB through PKC alpha and ROS in renal tubular epithelial cell. Renal tubular epithelial cells were exposed to 100μg/ml AOPP for 1h in the presence or absence of Go6983, Go6976, c-SOD, apocynin and DPI, and then the activation of AP-1 and NF-kB were assessed by western blot.14) AOPP activated RAS through PKC alpha and ROS in renal tubular epithelial cell. Renal tubular epithelial cells were exposed to 100μg/ml AOPP for 24h in the presence or absence of Go6983, Go6976, c-SOD, apocynin and DPI, and then AGT and AT1 protein levels were assessed by western blot.15) AOPP activated RAS through AP-1 and NF-kB in renal tubular epithelial cell. Renal tubular epithelial cells were exposed to 100μg/ml AOPP for 24h in the presence or absence of SN50 and Tanshinone IIA, then AGT and AT1 protein levels were assessed by western blot.3. Animal experiment1) Animal modelAll animal procedures were approved by the Animal Experiment Committee of Southern Medical University. Female waistar rats (initial weight,100 to 150g, Southern Medical University Animal Experiment Center) were fed standard rat chow ad libitum and given free access to water. The rats were subjected either to unilateral nephrectomy (UNX) or to sham operation (sham). One week after the operation, the UNX rats were randomized into 4 groups (n=6 in each group) matched for body weight and received the following treatment:1. intravenous injection of vehicle (PBS, ph7.4); 2. Intravenous injection of native RSA at 50mg/kg/d; 3. Intravenous injection of AOPPs-RSA at 50mg/kg/d; 4. Intravenous injection of AOPPs-RSA at 50mg/kg/day with intragastric administration of apocynin (Sigma,100mg/kg/d).All the treatments were given from randomization until the animals were sacrificed at week 3.At the end of week 3 after treatments, the animals (n=6) in each group were anesthetized with sodium pentobarbital and exsanguinated. The left kidneys were collected after perfusion with 50ml ice-cold normal saline. The 24-h urine samples were collected. Urinary albumin excretion was measured using an ELISA kit. Serum and urine creatinine levels, Serum albumin were determined using commercial kits. Mean systolic blood pressure by tail cuff method using the Softron BP system was measured before starting the treatment and at end of the study period.2) AOPP levels in plasma and renal tissue were measured by a spectrophotometer.3) AGT, ACE, AT1 mRNA levels in renal tissue were determined by RT-PCR.4) ACE, AT1 protein levels in renal tissue were determined by western blot;plasma and urinary AGT were measured by ELISA; ACE activity in serum and renal tissue was detected by a fluorospectrophotometer.5) Expression and location of AOPP, AGT, ACE, AT1 and angⅡin renal tissue were detected by immunohistochemistry.6) Activation of PKC alpha in renal tissue was determined by western blot.7) ROS production in renal tissue was measured by a fluorospectrophotometer.8) Membrane and cytosolic p47 phox, membrane p22 phox, Nox4, and Nox2 protein levels in renal tissue were determined by western blot.9) c-jun, c-fos protein levels and phosphoralytion of c-jun in renal tissue were determined by western blot10) Phosphoralytion of NF-kBp65 in renal tissue was determined by western blot.4. StatisticsAll data were expressed as mean±SEM. Continuous variables between groups were compared using one-way AN OVA, followed by LSD method when P≤0.05. Differences of the variables between two time points were determined by independent samples t test. Statistical analyses were conducted with SPSS 13.0 for Windows (SPSS, Chicago, IL). Significance was defined as P≤0.05.Results1. Cell part1) AOPP induced the activation of RAS in renal tubular epithelial cells. AOPP upregulation mRNA (P<0.001) and protein levels (P<0.001) of AGT, ACE, and AT1 in time and concentration dependent way. Moreover, AOPP increased angⅡconcentration (P<0.001) and ACE activity (P<0.001) in cell lysate and medium culture in a concentration and time dependent way.2) AOPP activated PKC alpha in renal tubular epithelial cells. The membrane phosphorylation of PKC alpha(ratio to total) was significantly increased in AOPP stimulated cells (P<0.001), while the phosphorylation of PKC zeta, delta, theta, epsilon(ratio to total) were unchanged after AOPP stimulation (P>0.05)3) AOPP induced the production of ROS in renal tubular epithelial cells. AOPP induced the production of ROS in a time and concentration dependent way (P<0.001). And the effects can be inhibited by the NADPH oxdise inhibitors DPI and apocynin (P<0.001), but can not be inhibited by other inhibitors (P>0.05).4) AOPP induced the activation of NADPH oxidase in renal tubular epithelial cells. AOPP induced the phosphorylation of p47phox (P<0.001), and binding of p22 phox with p47phox, Nox4 and Nox2 (P<0.001) in a time dependent way. Moreover, after long-time incubation with AOPP, the protein levels of p47phox,p22phox,Nox4和Nox2 were also upregulated (P<0.001).5) AOPP induced the activation of AP-1 in renal tubular epithelial cells. AOPP upregulated the protein expression of c-jun (P<0.001) and c-fos (P<0.001), and induced the phosphoralytion of c-jun (P<0.001) in a time dependent way.6) AOPP induced the activation of NF-kB in renal tubular epithelial cells. AOPP induced the phosphorylation of p65 (P<0.001) in a time dependent way.7) AOPP activated RAS through CD36 and RAGE in renal tubular epithelial cells. Western blot analysis indicated that megalin, CD36, SR-BI, Lox-1, galection-3, RAGE and SR-A were expressed in renal tubular epithelial cell. Inhibition CD36 and RAGE can significantly decrease the expression of AGT and AT1 in renal tubular epithelial cell (P<0.001)8) AOPP induced the activation of PKC alpha through CD36 and RAGE in renal tubular epithelial cells. Inhibition of CD36 and RAGE can significantly decrease the activation of PKC alpha in renal tubular epithelial cell (P<0.001).9) AOPP induced the activation of NADPH oxidase through CD36 and RAGE in renal tubular epithelial cells. Inhibition of CD36 and RAGE can significantly decrease activation of NADPH oxidase induced by AOPP (P<0.001).10) AOPP induced the production of ROS through CD36 and RAGE in renal tubular epithelial cells. Inhibition of CD36 and RAGE can significantly decrease the production of ROS indeced by AOPP (P<0.001).11) AOPP induced the NADPH oxidase activation and ROS generation through PKC alpha in renal tubular epithelial cells. Inhibition of PKC alpha can significantly decrease the NADPH oxidase activation and ROS generation induced by AOPP (P<0.001). Blockade the NADPH oxidase and ROS production partly inhibited the activation of PKC alpha induced by AOPP (P<0.001).12) AOPP induced the activation of AP-1 and NF-kB through PKC alpha and ROS in renal tubular epithelial cells. Inhibition of PKC alpha and ROS can significantly decrease the activation of AP-1 and NF-kB induced by AOPP (P<0.001).13) AOPP activated RAS through PKC alpha and ROS in renal tubular epithelial cells. Total PKC inhibitor Go6983, PKC alpha inhibitor Go6976, c-SOD, apocynin and DPI can significantly reduce the upregulation of AGT and AT1 induced by AOPP(P<0.001).14) AOPP activated RAS through AP-1 and NF-kB in renal tubular epithelial cells. NF-kB inhibitor SN50, AP-1 inhibitor TanshinoneⅡA can significantly reduce the upregulation of AGT and AT1 induced by AOPP (P<0.001).3. Animal part1) Characteristics of the Experimental RatsAll rats survived. UNX rats showed significantly greater kidney weight as compared with control rats (P<0.001). Intervention of the NADPH oxidase inhibitor apocynin significantly reduced kidney weight in AOPP-treated UNX rats as compared with the animals received AOPPs challenge in the absence of apocynin (P<0.001). Mean systolic blood pressure and body weight was comparable among all the groups (P>0.05)2) AOPP levels in plasma and renal tissue.The concentration of plasma AOPP increased by approximately one-fold in AOPPs-RSA-treated UNX rats at week 3 compared with vehicle-or RSA-treated UNX animals(P<0.001). In contrast, there was no significant difference in plasma albumin levels among groups (P>0.05). Similarly, AOPP levels in the renal homogenates(P<0.001) and immunostaining of AOPP in renal tissue increased in UNX rats. Intervention of apocynin decreased the AOPP levels in both plasma and renal tissue (P<0.001).3) AOPP activated RAS in renal tissue. Compared with sham, vehicle-or RSA-treated UNX animals, AGT, ACE, AT1 mRNA and protein levels in AOPP-treated UNX kidney were significantly increased (P<0.001). Meanwhile, urinary AGT, renal angiotensinⅡconcentration, renal ACE activity (P<0.001) and immunostaining of AGT, ACE, AT1 and AngⅡwere also upregulated in AOPP-treated UNX rats. Intervention of apocynin significantly decreased the AOPP-induced RAS upregulation in renal tissue (P<0.001). Plasma AGT, angiotenisnⅡand serum ACE activity were comparable among all the groups (P>0.05)4) AOPP activated PKC alpha in renal tissue. Compared with sham, vehicle-or RSA-treated UNX animals, membrane phosphorylated PKC alpha(ratio to total) was significantly increased in AOPP-treated UNX kidney (P<0.001). Intervention of apocynin significantly decreased the AOPP-induced PKC alpha activation in renal tissue (P<0.001)5) AOPP activated NADPH oxidase in renal tissue. Compared with sham, vehicle-or RSA-treated UNX animals, the ROS production in AOPP-treated UNX kidney was significantly increased (P<0.001). Intervention of apocynin significantly decreased the AOPP-induced ROS production in renal tissue (P<0.001).To verify the role of NADPH oxidase, we study the expression of NADPH oxidase subunits in renal tissue. Compared with sham, vehicle-or RSA-treated UNX animals, membrane p47phox, p22phox, Nox4, and Nox2 were upregulated in AOPP-treated UNX kidney (P<0.001), while cytosolic p47phox was unchanged among all the groups (P>0.05). Intervention of apocynin significantly decreased the AOPPs-induced these changes in renal tissue (P<0.001)6) AOPP activated AP-1 in renal tissue. Compared with sham, vehicle-or RSA-treated UNX animals, expression of c-jun and c-fos, and phosphoralytion of c-jun were significantly increased in AOPP-treated UNX kidney (P<0.001) Intervention of apocynin significantly decreased the AOPP-induced these changes in renal tissue (P<0.001)7) AOPP activated NF-kB in renal tissue. Compared with sham, vehicle-or RSA-treated UNX animals, the phosphoralytion of p65 were significantly increased in AOPP-treated UNX kidney (P<0.001). Intervention of apocynin significantly decreased the AOPP-induced phosphoralytion of p65 in renal tissue (P<0.001)4. Conclusion:1. Cell partAOPP induced the activation of RAS likely through a CD36, RAGE-mediated signals involving PKC alpha, NADPH oxidase dependent ROS generation, activator protein-1(AP-1) and nuclear transcription factorκB (NF-κB) in cultured renal tubular epithelial cells.2. Animal part1. AOPP activated RAS in renal tissue. Intervention of apocynin significantly decreased the AOPP-induced RAS activation in renal tissue.2. AOPP activated renal PKC alpha. Intervention of apocynin significantly decreased the AOPP-induced phospholyration of PKC alpha in renal tissue.3. AOPP activated renal NADPH oxidase-dependent ROS production.4. AOPP activated AP-1 in renal tissue.5. AOPP activated NF-κB in renal tissue.In conclusion, Our data indicated that AOPP induced the activation of RAS likely through a CD36, RAGE-mediated signal involving PKC alpha, NADPH oxidase dependent ROS generation, activator protein-1 (AP-1) and nuclear transcription factorκB (NF-κB).
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