| BackgroundRenal interstitial fibrosis, characterized by disappearance of peritubular capillaries, interstitial inflammatory cell infiltration, massive interstitial myofibroblast activation and excessive matrix proteins accumulation, is the final common pathway of virtually all kinds of progressive kidney diseases leading to end-stage renal disease (ESRD). Renal interstitial fibrosis involves immune inflammatory response, renal inherent cells apoptosis, oxidative stress reaction enhancement and fibroblasts proliferation and activation and epithelial cells to fibroblasts. Oxidative stress reaction enhancement plays an importent role in renal interstitial fibrosis.Oxidative stress refers to a serious imbalance between the in vivo or intracellular oxidation system and antioxidant defense system. Mounting evidence has established a crucial role of oxidative stress in the progressive of renal interstitial fibrosis. Sedeek et al pointed out that in pathologic conditions, a surplus of ROS in tissue results in oxidative stress with various injurious consequences such as inflammation and fibrosis. Studies have shown that mitochondria are energy-producing organelles which constitute the primar cellular source of ROS and are the primary target of oxidative damage. Mitochondria oxidate damage induceed mitochondrial dysfunction, which is closely related to cancer, aging, Parkinson’s disease, alzheimer’s disease, ischemic hypoxic brain damage, fatty liver disease, diabetes, septic-shock, myocardial ischemia-reperfusion injury and other diseases. Mitochondrial dysfunction results in ATP consumption, excessive ROS production,the release of apoptosis factor such as cytochrome C (Cyto C) and DNA fractions,promoting apoptosis through DN or protein Oxidative stress.,then inducing inflammation and fibrosis. Kidney is a high energy metabolic organs, riching in mitochondria, mitochondrial dysfunction may play a key role in renal interstitial fibrosis. Granata et al. argued that that patients with chronic kidney disease (CKD) 2-3 stages may have an impaired mitochondrial respiratory system and this condition may be both the consequence and the cause of an enhanced oxidative stress. Recently, studies have pointed out that dysfunctional mitochondria participate in the progression of renal fibrosis in CKD. Pirfenidone and peroxisome proliferator-activated receptor y (PPARy) may be effective on the onset of renal fibrosis by protection of mitochondria. Oxidative stress is a very factor to induce mitochondrial dysfunction. Mitochondrial dysfunction induced by oxidative stress activates the mitochondrial apoptotic pathways leading to renal intrinsic cell apoptosis. A large number of previous studies have confirmed the number of tubular cells apoptosis was positively correlated to renal interstitial fibrosis, and it has been suggested that inhibition of tubular cell apoptosis may protect against macrophage infiltration and interstitial fibrosis. These data indicated that mitochondrial dysfunction induced by oxidative stress and renal tubular epithelial cells apoptosis are closely related to renal fibrosis.Hypochlorite-modified albumins (HOC1-alb) are markers of oxidized protein. They are also a new class of mediators promoting oxidation and inflammation, correlating with the progression of Atherosclerotic lesions, diabetic nephropathy and CKD. In the remnant kidney model, HOC1-alb accumulation promotes renal fibrosis and detedorates renal dysfunction, probably via a redox-sensitive inflammatory pathway. However, as oxidative stress induced factors, whether HOC1-alb can induce mitochondrial oxidative stress injury and dysfunction, activate mitochondrial apoptosis pathway leading to renal tubular epithelial cells apoptosis and further worsen renal fibrosis remains unknown.SS peptide is a small molecule polypeptide, which is composed of four amino acid residues, has been confirmed containing a particular domain sequence enable it selectively to target and concentrate 1,000 to 10,000-fold in the inner mitochondrial membrane not relaying on mitochondrial membrane potential. Therefore, it can play an effective antioxidant effect against mitochondria oxidate damage. SS-31 has played its efficient antioxidant role in ischemic brain injury, diabetic retinopathy, alzheimer’s disease. However,its protective effect and mechanism on the renal interstitial fibrosis is still not sure.Unilateral ureteral obstruction (UUO) is a mature experimental model of apoptosis and fibrosis. The present stuady was conducted to test the hypothesis that HOCl-alb induce mitochondrial oxidative stress injury and dysfunction, activate more renal tubular epithelial cells apoptosis and further accelerate renal interstitial fibrosis.Methods1. HOC1-RS A preparation and determination in vitroRat serum albumin was exposed to HOCI (1:140 molar ratios) for 30 minutes at room temperature. The products were dialyzed overnight against PBS to remove any free HOCI and then sterilzed using 0.22 μm micropore filter membrane.and conserved at-80 degree. To detect the content of HOC1-RSA, different concentrations of chloromine-T is used to make criteria curve. The absorbance of the reaction mixture at 340 nm is immediately read in a microplate reader. Endotoxin levels in the preparation were tested with limulus amebocyte lysate kit (Sigma) and were found to be less than 0.025 endotoxin units/ml. The content of HOC1-RSA was 5.3±0.4 nmol/mg protein versus 0.2±0.03 nmol/mg protein in unmodified RSA.2. SS peptide synthesis and determination in mitochondriaa. SS peptide synthesisSS petides were synthesised by company in HangZhou, and the sequences of each SS peride was as follow:SS-02 (Dmt-D-Arg-Phe-Lys-NH2, Dmt=2’,6’-dimethyltyrosine), SS-31 (D-Arg-Dmt-Lys-Phe-NH2).b. Mitochondria extractedIsolation of mitochondria from kidney cortexes according the kit instructionc. Identification SS peptide entering the mitochondria in vitroUsing [3H] to tag SS-02, using liquid scintillation counter to location SS-02 in mitochondria. Results show that the SS-02 distributed in the mitochondrial outer membrane about 20-30%, about 50% in mitochondrial inner membrane, and 20-30% in mitochondrial matrix.3.Animals treatmentThe unilateral ureteral obstruction rats were served as animal model. Rats were randomly assigned to five groups:(1) Sham group; (2) UUO+normal saline (NS): UUO rats received normal saline once daily; (3) UUO+RSA group:RSA 100 mg/kg, iv.once daily; (4) UUO+HOC1-RSA group:HOC1-RSA100 mg/kg, iv.once daily; (5) UUO+HOC1-RSA+SS-31 group:100 mg/kg, iv.once daily; SS-313 mg/kg ip once daily.4. Animals were sacrificed at day 14. Plasma and renal tissue samples were collected.a. Mitochondrial damage assay(1) Electron microscopic analysis technique to detect ultrastructure of mitochondria in renal cortex,(2) Isolation of mitochondria and cytosolic fractions using differential velocity centrifugation; using 5,-5’,-6,6’-tetrachloro-l,-l-’,-3,-3-’-tetrethyl benzimidalylcarbocyanine iodide (JC-1) to measure mitochondrial membrane potential (△Ψm),(3) Isolated mitochondria reactive oxygen species (ROS) measurement using 2’,-7’-dichlorodihydrofluorescein diacetate (DCFDA),(4) Using the DNeasy tissue kit to extract total DNA from kidney cortexes, using Real-time PCR to detect mitochondrial DNA (mtDNA) copy number,(5) Using ATP Colorimetric/Fluorometric Assay Kit to detect the ATP content.b. Oxidative stress assay(1) Using spectrophotometer method to detect kidney homogenate HOCl-alb,(2) Lipid peroxides in the renal conical homogenate were measured as thiobarbituric acid reactive substances (TBARS) by fluoromctric assay,(3) Mn-SOD activity assays were performed according to the manufacturer’s instructions(Beyotime Bioengineering Institute, Nanjing, China).c. Apoptosis assay(1) Using TdT-mediated dUTP nick end labeling (TUNEL) Assay to detect renal tubular epithelial cell apoptosis,(2) Western-blot for mitochondria and cystosol cytochrome C, cleaved caspase-3/9/7 and PARP-1.d. Interstitial fibrosis assay(1) MASSON staning for collagen,(2) Real-time PCR, Western-blot and immunohistochemical method to detect Collagen-I、α-SMA、Fibronectin.StatisticsAll values are presented as mean±SD. The significance of differences among mean values was determined by One-way ANOVA. Pirwise comparisons were evaluated using the LSD procedure when two groups of data had equal variances,if not the Dunnett’s T3 procedure was used. Statistical analyses were conducted with SPSS 20.0. The accepted level of significance was P<0.05.Result1. Effect of HOC1-alb on mitochondrial function(1) Electron microscopy results showed that compared sham, mitochondria in kidney cortex from the UUO rats exhibited different degree of swelling, disrupted cristae architectures and even disappeared,the flameng socres of mitochondria were higher compared with sham (P<0.05), HOC1-RSA administration exhibited more-damaged cristae structures and even disappeared compared with other UUO groups (P<0.05), SS-31 treatment ameliorated the damaged mitochondria caused by HOC1-RSA administration and UUO surgery(P<0.05).(2) MMP in kidney cortexes from four groups UUO rats deceased drastically compared to the sham group (P＜0.05), HOC1-RSA administration exhibited lower MMP compared with other UUO groups (P＜0.05), A significant increase in MMP was observed after SS-31 treatment compare to UUO+HOC1-RSA, UUO+NS, and UUO+RSA group (P＜0.05).(3) Isolated mitochondria from kidney cortexes in four UUO groups exhibited a dramatic increase in ROS production compared with sham, HOC1-RSA administration significantly elavated ROS production compared with other groups (P＜0.05), and SS-31 treatment significantly reduced ROS production compared to UUO+HOC1-RSA, UUO+NS, and UUO+RSA group (P＜0.05).(4) ATP levels in kidney cortexes from four UUO groups were dramatically lower than sham levels (P＜0.05), HOC1-RSA administration exhibited lower ATP levels compared with other UUO groups (P＜0.05), and SS-31 treatment significantly improved ATP levels compare to UUO+HOC1-RSA, UUO+NS, and UUO+RSA group (P＜0.05).(5) The mtDNA copy numbers in kidney cortexes from four UUO groups were significantly decreased compared with sham groups (P＜0.05), HOC1-RSA administration futher decreased mtDNA copy numbers compared to other UUO groups (P<0.05), and these numbers improved with SS-31 treatment compared to UUO+HOC1-RSA, UUO+NS, and UUO+RSA group (P<0.05).2. Effect of HOC1-alb on oxidative stress(1) Serum and kidney homogenate HOC1-alb from four UUO groups were both increased significantly as compared with the sham group (P＜0.05), HOC1-alb administration increased the levels of HOC1-alb in serum and kidney homogenate as comcared with other UUO groups (P＜0.05), and SS-31 treatment significantly decreased HOC1-albs levels as compared to UUO+HOC1-RSA, UUO+NS, and UUO+RSA group (P<0.05).(2) The TBARS in the renal homogenate from four UUO groups indicates reduced antioxidant capacity were notably enhanced compared with sham group (P＜0.05), HOC1-RSA administration futher enhanced TBARS levels compared to other UUO groups (P＜0.05), and SS-31 treatment significantly decreased TBARS levels compared to UUO+HOC1-RSA, UUO+NS, and UUO+RSA group (P＜0.05).(3) Mn-SOD activity was decreased in four UUO groups rats compared with sham group (P＜0.05), HOC1-RSA administration futher decreased Mn-SOD activity compared to other UUO groups (P＜0.05), and SS-31 treatment significantly improved Mn-SOD activity compared to UUO+HOC1-RSA, UUO+NS, and UUO+RSA group (P＜0.05).3. Effect of HOC1-alb on renal tubular cells apoptosis(1) The obstructed kidney from four UUO groups showed significantly higher tubular apoptosis as compared with sham group (P＜0.05), HOC1-RSA administration futher increased the number of tubular apoptosis compared to other UUO groups (P＜0.05), SS-31 treatment significantly decreased the number of tubular apoptosis compared to UUO+HOC1-RSA, UUO+NS, and UUO+RSA group (P＜0.05).(2) Cytochrome C decreased significantly in the mitochondrial fractions and increased significantly in the cytosolic fractions from four UUO group rats compared to the sham group,which suggested mitochondrial leakage in UUO rats (P＜0.05), HOC1-RS A administration futher aggravated mitochondrial leakage as compared with other UUO groups (P＜0.05), SS-31 treatment significantly prevented mitochondrial leakage compared compared to UUO+HOC1-RSA, UUO+NS, and UUO+RSA group (P＜0.05).(3) Cleavage of caspase-3、caspase-7、caspase-9 and PARP-1 levels were significantly increased in kidney cortexs from four UUO group rats as compared with sham group, HOC1-RSA administration futher promoted the cleavage of caspase-3 caspase-7、 caspase-9、 PARP-1 as compared with other UUO groups (P＜0.05), SS-31 treatment significantly decreased cleavage of caspase-3、 caspase-7、 caspase-9 and PARP-1 levels compared to UUO+ HOC1-RSA, UUO+NS, and UUO+RSA group (P＜0.05).4. Effect of HOC1-alb administration on renal fibrsis(1) MASSON staining indicated that no histologic abnormalities of the kidneys were observed in sham group, fibrosis could be detected in renal interstitial area from four UUO group rats, HOC1-RSA administration aggravated renal fibrosis as compared with other UUO groups, SS-31 treatment decreased renal fibrosis as compared with UUO+HOC1-RSA, UUO+NS, and UUO+RSA group.(2) Collagen-I、Fibronectin、 α-SMA expression assessed by Western blot and real-time PCR in kidney cortexs in rats from four UUO groups significantly increased as compared with sham group (P<0.05), HOC1-RSA administration futher increased Collagen-I、 Fibronectu、 α-SMA expression as compared with other UUO groups (P<0.05), SS-31 treatment decreased significantly increased Collagen-I、 Fibronectim a-SMA expression compared to UUO+HOC1-RSA, UUO+NS, and UUO+RSA group (P＜0.05).The results of immunohistochemical staining were consistent with the results of Western blot and real-time PCR.Conclusiona.Kidney in UUO rat showed significantly increased markers of oxidative stress, mitochondrial structure and function impaired, renal interstitial cells apoptosis and renal interstitial fibrosis.b. HOC1-alb is a known oxidative stress promoting medium.Repeated injections of HOC1-alb aggravated pathological effects in UUO rat, and SS-31 treatment ameliorated the damaged caused by HOC1-RSA administration and UUO surgery. This reveals that mitochondrial oxidative stress injury participates in renal interstitial fibrosis.Mitochondrial oxidative stress injury and dysfunction is a new intervention target in renal interstitial fibrosis. Mitochondrial targeting antioxidants (SS-31) might be a new drug in protecting renal interstitial fibrosis. |
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