| BackgroundAccording to the report in 2014 estimated that the global prevalence of diabetes was 8.3%, affecting more than 387 million adults worldwide. The figure will rise to 55% involving more than 592 million patients in the end of 2030.Diabetic mellitus (DM) and its complications have become one of the most important diseases that harm to human health, and diabetic nephropathy (DN) is one of the chronic complications of diabetes which is the most common and serious. DN is also the main cause of death within the scope of the global end of end-stage renal disease (ESRD) which serious impact on the quality of human life. Although clinical studies have shown that elevated blood glucose is the primary risk of diabetic complications and development factor, but just effectively control blood glucose couldn’t prevent the complications completely. In recent years, oxidative stress is considered to be the reason of the early event in the pathogenesis of DN and important common mechanisms. The role of glomerular podocytes damage caused by oxidative stress in the progression of DN gets more and more attention. Therefore, reducing mitochondrial ROS production in the role of disease prevention and control has attracted more and more attention.Oxidative stress is caused by the imbalance between oxidation and antioxidant. Under the normal physiological conditions, body can produce a small amount of ROS (reactive oxygen species) in normal metabolism, and also exists scavenging free radicals and inhibiting free radical reaction system, through which the free radicals are cleared. So ROS generation and removement keep in a state of dynamic equilibrium. If the dynamic balance is destroyed, it causes ROS to accumulate in the body, causing molecules, cells and body injury. The pathological process including is oxidative stress. The process of oxidative stress produce large lipid and protein oxidation product, activity of aldehydes, oxidized sulfhydryl, hydroxyl compounds.Early manifestations of DN include increased glomerular hyperfiltration and urinary albumin excretion, glomerular hypertrophy, basement membrane thickening, extracellular matrix accumulation and mesangial hyperplasia. As the progression of the disease, large amounts of protein appear in the urine, then glomerular and interstitial sclerosis, and ultimately the development of ESRD. Previous researches most focused on the mesangial injury that the increase of mesangial matrix was the core lesion of DN. However, in recent years more and more studies suggest that podocytes injury plays a crucial role in the early stage of DN. It is of great significance in the integrity of the filtration barrier, the production of proteinuria and glomerular sclerosis.Mitochondria are the main parts of ROS generation and also the most sensitive to oxidative stress reaction. Kidney is a high energy metabolic organ with abundant mitochondria. Mitochondrial dysfunction may play a key role in renal interstitial fibrosis, which has been widely concerned in recent years.In mitochondria, cytochrome C is immobilized on the inner membrane of mitochondria through combined with the anionic phospholipid. Myocardial phospholipid only exists in mitochondria and is found in the inner mitochondrial membrane which plays the role of maintaining membrane fluidity and stability of mitochondria. The molecular link between myocardial phospholipid and cytochrome C include electrostatic interaction of cytochrome CA terminal, hydrophobic interaction and hydrogen bonding effect of C terminal. When electrostatic interaction and hydrophobic interaction between cytochrome C and myocardial phospholipid was destroyed, cytochrome C woule leave mitochondria and flow into the cytoplasm. Previous discovery found that myocardial phospholipid oxidation reduced its affinity for cytochrome C, and recently study showed that the oxidative regulation of myocardial phospholipids promoted the binding of cytochrome C to the mitochondrial inner membrane. The release of cytochrome C in the apoptosis process is divided into two steps. The first step is the separation betweet cytochrome C and myocardial phospholipid and then the release of cytochrome C into the cytoplasm through the penetration effect of mitochondrial outer membrane. More and more data showed that ROS promoted the separation between myocardial phospholipid and cytochrome C, and then cytochrome C passed through the open pores punched by pro-apoptotic protein on the mitochondrial outer membrane into the cytoplasm. The study on myocardial ischemia of reperfusion rabbit heart also revealed the increased of ROS led to the oxidation and reduction of myocardial phospholipids with the decrease of COX activity in mitochondria. In addition, cytochrome C released from mitochondria in an ROS dependent mode in the model of glutamate toxicity in neurons. Bcl-2 can regulate the permeability of some apoptotic protein precursor in the mitochondrial membrane. Bax located upstream of the mitochondria can allow some ions and small molecules such as cytochrome c across the mitochondrial membranes into the cytoplasm, and further activate the caspase cascade, resulting in cell apoptosis. Experiments showed that cytochrome C released from mitochondria was a critical step in apoptosis, and combined with caspase-9 to form apoptotic bodies, and the activation of caspase-9 can activate other caspases such as caspase-3 and caspase-7. Among them, caspase-3 is the key apoptosis executive in the caspase cascade reaction.Traditional antioxidants have poor efficacy, mostly because these compounds are distributed in a large number of cells or cytoplasm, can not enter the mitochondria, it is difficult to play the role of antioxidant. SS-31 belongs to the small molecule peptide family which can penetrate cells, capable of targeting and aggregation in mitochondrial membrane. SS-31 can scavenge intracellular ROS and inhibition of mitochondrial ROS production, thereby preventing the change of mitochondrial permeability and the release of cytochrome C. Studies have shown that to retinal damage in diabetic rats, SS-31 can effectively improve the retinal oxidative stress and protect the integrity structure of retina.HOCl-alb is a protein oxidative modification product formed during oxidative stress and was isolated for the first time from uremic patients by Witko-Sarsat and his colleagues in 1996. It was confirmed to be an aggregate of albumin likely resulting from disulfide bridges and/or dityrosine cross linking. In vitro, the interaction between HOCl and albumin can produce HOCl-modified albumin with the same biochemical characteristic as that extracted from uremic patients. HOCl-alb is not only a product of oxidative stress and a marker, it has been studied as a strong pro-inflammatory reaction medium. HOCl-alb could induce the macrophage chemotaxis, MCP-1 and TGF-expression, the increase of ROS, proteinuria and glomerular pathological changes aggravated in diabetic rats. But it is not clear whether it is able to induce or aggravate podocytes apoptosis through mitochondrial signaling pathways in diabetic nephropathy.Our research using diabetic rats induced by STZ to verify our assumption that HOCl-alb induces podocyte mitochondrial dysfunction and intracellular ROS, activation of the apoptotic signal, induces apoptosis of podocytes, resulting in impaired glomerular basement membrane barrier function, eventually leading to proteinuria and glomerulosclerosis in diabetic environment. Mitochondrial targeting antioxidant SS-31 may inhibit the apoptosis of the podocytes by blocking the oxidative stress damage of mitochondria, and prevent or delay the development of DN.Materials and methods1. HOCl-alb-RSA preparation and identification:HOCl-alb-RSA was prepared in vitro as described previously. Equivalent volumes of RSA and HOCl were mixed for 30 min at room temperature, and HOCl-alb-RSA was prepared in molar ratios of 1:140. A mixture of equivalent volumes of RSA and PBS was used as the control. The prepared HOCl-alb-RSA and unmodified RSA without modification were dialyzed overnight against PBS to remove any free HOCl, sterilized by filtration through a 0.22μm microporous membrane, and stored at 4℃. All glassware in the experiment was baked at 180 degrees for 4 hours to deal with endotoxin.The HOCl-alb-RSA content was measured as the absorbance at 340 nm under acid conditions, based on a chloramine-T standard. The content of HOCl-alb in the stock HOCl-alb-RSA preparation was 5.14±0.25nmol/mg protein versus 0.21±0.05nmol/mg protein in unmodified RSA. A limulus test showed that the content of endotoxin in both preparations was< 0.25 EU/ml.2. Synthesis and identification of SS peptide into mitochondria2.1 Synthesis of SS peptideSS peptide was synthetized with the assist of the Company Zhongtai in Hangzhou. SS peptide amino acid sequence and chemical structure:SS-02 (Dmt-D-Arg-Phe-Lys-NH2, Dmt=2’,6’-dimethyltyrosine), SS-31 (D-Arg-Dmt-Lys-Phe-NH2).2.2 Mitochondrial extractionExtract mitochondria according to kit instructions with renal tissue (cortex).2.3 Identification of SS peptide into mitochondria in vitroSynthesis of SS-02 by solid phase method(Dmt-D-Arg-Phe-Lys-NH2, Dmt=2’,6’-dimethyltyrosine), mark SS-02 with [3H], and then using liquid scintillation counter to detect the localization of SS-02 in mitochondria. About 20-30% SS-02 distributed in mitochondrial outer membrane, about 50% distributed in mitochondrial inner membrane,and 20-30% in mitochondrial matrix.3. Experimental animal protocol and grouping3.1 Experimental animal protocol48 adult male Sprague-Dawley rats (purchased in the Southern Medical University Animal Experiment Center, initial weight 180-242g) were maintained under standardized conditions and fed with standard rodent chow in Southern Medical University SPF experimental animal center. After fasting 16 hours, a single intraperitoneal injection of streptozotocin (STZ) 60mg/kg (dissolved in 0.01 mol/L citric acid buffer, pH4.5). After 72 hours, the blood glucose meter was used to measure the tail blood glucose. Rats with blood glucose> 16.7mmol/L was used as an animal model of diabetes mellitus (DM).3.2 Rats were randomly assigned to six groups.1. Normal control group 1:PBS, iv. once daily;2. Normal control group 2:RSA 40mg/kg, iv. once daily;3. DM rats RSA group:RSA 40mg/kg, iv. once daily;4. DM rats HOCl-alb group:HOCl-alb-RSA 40mg/kg, iv. once daily;5. DM rats SS-31 group:3mg/kg SS-31, ip. once daily;6. DM rats HOCl-alb and SS-31 group:HOCl-alb-RSA 40mg/kg, iv. once daily; 3mg/kg SS-31, ip. once daily;3.3 Obtain specimensRats were sacrificed at week sixteen. Rats were placed into metabolic cages to collecte 24 hours urine one day before being sacrificed. The urine was centrifuged 20min (4℃ 3000rpm), and frozen in -70℃ refrigerator after packing. The rats were fully anesthetized and fixed on the rat board. The abdominal aortic blood was taken with EDTA-K2 tube and centrifuged 15min (4℃ 3000rpm)within 30 min to collect the plasma, then frozen in -70℃ refrigerator after packing.4℃ saline perfusion of abdominal aorta to remove the red blood cell cycle, until the kidey was pale. Remove the left and right kidneys rapidly, and then separate the renal cortex and medulla.4. Detection index4.1 Biochemical index:Automatic biochemistry analyzer was used to measure the blood glucose (Glu), albumin (ALB), urinary microalbumin (mAlb), serum creatinine (SCR), urine creatinine (UCR), calculated creatinine clearance rate (CCR) (ml/min/ kg)= urine creatine/creatinine 24 (1440min) x 24 hour urine volume/rat body weight.4.2 Renal homogenate and plasma levels of HOCl-alb were determined as described above.4.3 Urinary 8-OHdG excretion was detected by an ELISA kit.4.4 Histopathological observation:Cortex was embedded in paraffin, cut in 4μm sections. Then slides were stained by use of PAS and Masson.4.5 Immunostaining to detect podocyte numbers:Cortex was embedded in paraffin,. Then slides were stained by use of TUNEL kit to count podocyte numbers.4.6 Western Blotting to detect apoptotic protein:Collect total protein to detect the expression of caspase-3, caspase-7, caspase-9, and PARP-1. Collect mitochondrial and cytoplasmic protein of the renal tissue to detect the expression of Cytochrome C.Results1. The physical and biochemical changes in rats1.1 Body weightsThe weights were significantly reduced in DM versus controls ①,②(① 623.72±21.20, ② 619.23±23.47 vs ③ 249.62±29.11, ④ 173.35±27.60, ⑤ 310.14±32.75, ⑥241.38±27.29,P<0.001). Injection of HOCl-alb and SS-31 treatment had no significantly effect on weights in DM (P>0.05 vs ③)1.2 Blood glucoseBlood glucose were higher in DM versus controls ①,②(①5.6±0.8, ②5.8±1.0 vs ③29.78±2.51, ④31.46±3.23, ⑤32.34±2.76, ⑥30.40±2.48, P<0.001). Injection of HOCl-alb and SS-31 treatment had no significantly effect on blood glucose in DM (P>0.05vs③)1.3 Urinary protein excretion of diabetic ratsIn diabetic rats,24 hour urinary protein is higher versus controls ①,② (① 0.19±0.05, ②0.21±0.03 vs ③0.34±0.09,④0.51±0.10,⑤0.27±0.08,⑥0.30±0.11, P<0.05).Injection of HOCl-alb increased urinary protein excretion in diabetic rats (③0.34±0.09 vs ④0.51±0.10, P< 0.01). SS-31 treatment significantly reduce urinary protein excretion induced by HOCl-alb in diabetic rats (④0.51±0.10 vs ⑤ 0.27±0.08, ⑥0.30±0.11, P< 0.01)1.4 Creatinine clearance rateCreatinine clearance rate were higher in DM versus controls ①,② (①6.24±1.09, ②6.37±1.22 vs ③10.61±2.36, ④13.95±4.62, ⑤7.67±1.84, ⑥8.80±2.03, P<0.05). Injection of HOCl-alb increased serum creatinine clearance rate in diabetic rats (③ 10.61±2.36 vs ④13.95±4.62, P< 0.05). SS-31 treatment ameliorated the increase of serum creatinine clearance rate induced by HOCl-alb (④13.95±4.62, ⑤7.67±1.84, ⑥8.80±2.03, P<0.01)2. Pathological changes in kidneyThrough light microscope observation of renal tissue Masson staining and PAS staining, we found that the glomerular structure was complete, uniform size, renal tubulars were smooth and there was no proliferation of basement membrane and mesangial cell in the normal control group. The glomerular volume increased, glomerular basement membrane thickening and mesangial proliferation, and the renal tubular epithelial cells showed focal necrosis, granular and vacuole degeneration in diabetic rats. The pathological changes of diabetic rats in group HOCl-alb were more serious than those in group RSA rats. Most pathological changes in SS-31 group of diabetic rats were blocked by injection of mitochondrial targeting peptide SS-31. These results suggested that mitochondrial targeting peptide SS-31 treatment reduced the early inflammatory reaction induced by HOCl-alb.3. Changes of oxidative stress index3.1 The level of HOCl-alb in kidney homogenateIn diabetic rats kidney homogenate, the level of HOCl-alb were higher versus controls ①,② (①5.67±0.19, ②6.75±0.37 vs ③16.32±1.07, ④20.25±1.02,⑤ 9.38±0.75,⑥15.71±0.38, P< 0.05). And injection of HOCl-alb increased the level of HOCl-alb (③16.32±1.07vs ④20.25±1.02, P< 0.01). SS-31 treatment alleviated the level of HOCl-alb induced by HOCl-alb (④20.25±1.02 vs ⑤9.38±0.75,⑥ 15.71±0.38, P<0.01)3.2 The level of HOCl-alb in plasmaPlasma HOCl-alb in diabetic rats were higher versus controls ①, ② ( ① 85.34±11.28, ②100.63±14.7 vs ③193.54±10.69,④426.67±14.94, ⑤143.33±4.39, ⑥237.5±12.16, P<0.05). Injection of HOCl-alb elevated the level of HOCl-alb (③ 193.54±10.69 vs ④426.67±14.94, P<0.01). SS-31 treatment alleviated the level of plasma HOCl-alb induced by HOCl-alb (④426.67±14.94 vs ⑤143.33±4.39,⑥ 237.5±12.16, P<0.01)3.3 Urinary 8-OHdG quantificationWe found that 24 hours urine 8-OHdG quantitative in diabetic rats were increased versus controls ①,② (①52.88±13.45,②58.56±20.65 vs ③170.83±23.29,④ 473.2±66.69, ⑤93.77±48.9, ⑥132.86±54.18, P<0.05). Injection of HOCl-alb elevated urinary 8-OHdG in diabetic rats (③170.83±23.29 vs ④473.2±66.69, P< 0.01). SS-31 treatment alleviated the level of urinary 8-OHdG induced by HOCl-alb (④473.2±66.69 vs ⑤93.77±48.9, ⑥132.86±54.18, P<0.01)4. Changes of Cyt C expressionThe expression of Cyt C was detected by Western Blotting. The results showed that the expression of cytoplasmic Cyt C was up-regulated in diabetic rats compared with normal control rats (P<0.015 vs group①、②). The level of cytoplasmic Cyt C in HOCl-RSA-treated diabetic rats was further increased compared with diabetic rats (P<0.008 vs group③). SS-31 intervention could ameliorate the level of cytoplasmic Cyt C induced by HOC1-RSA (P<0.021 vs group④) Changes of cytoplasmic Cyt C in each group were contrary to cytoplasmic Cyt C.5. Changes of podocyte apoptosisIn order to detect whether HOCl-alb promoted podocyte apoptosis, we used fluorescence labeling of TUNEL to detect apoptosis of podocytes. In HOC1-RSA-treated group, TUNEL positive cells was significantly higher than RSA-treated diabetic rats (p=0.012). Mitochondrial targeting peptide SS31 treatment significantly reduced podocyte apoptosis induced by HOCl-alb (p= 0.023). The SS31 could reduce oxidative stress in podocytes, thereby reducing the podocyte apoptosis.6. The expression of podocyte apoptosis proteinIn the signal pathway of apoptosis induced by oxidative stress, caspase-3 is the most critical apoptosis executor in the downstream of caspase. The expression of caspase-3, caspase-7, caspase-9, and PARP-1 were detected by Western Blotting. The results showed that caspase-3, caspase-7, caspase-9, PARP cleavage products expression were significantly increased in HOCl-alb-treated group (P< 0.005vs group ③, and these cleavage fragments expressed significantly reduced in SS-31 treatment diabetic rats (P< 0.019 vs group④)7. Changes in the number of podocytesIn order to verify HOCl-alb induced podocyte apoptosis leads to glomerular podocyte loss, thereby reducing the number of glomerular podocytes, immunostaining was used to detect podocyte numbers. The results showed that in HOCl-alb-treated group, the number of positive podocyte is significantly lower than RSA-treated diabetic rats (P<0.011vs group③). Mitochondrial targeting peptide SS-31 treatment significantly increased the loss of podocytes induced by HOCl-alb (P<0.009 vs group④)Use Western Blotting to detect the expression of WT-1 protein. The results showed that WT-1 expression was significantly decreased in HOCl-alb-treated group compared with RSA group (P<0.003 vs group③). Mitochondrial targeting peptide SS-31 treatment significantly increased the expression of WT-1 (P< 0.007 vs group ④).Conclusion1. Administration of HOCl-alb could aggravate renal injury in diabetic rats, induce apoptosis of glomerular podocytes. The pathway may be oxidative stress induced damage of mitochondria, change of mitochondrial permeability and efflux of cytochrome C. The activation of cytochrome C in cytoplasm induced the up-regulation of procaspase-3, procaspase-7, procaspase-9 and then activated the PARP-1 cleavage products. These apoptotic proteins triggered apoptosis pathway. Podocyte apoptosis resulted in podocyte loss, and cause the reduction in the number of glomerular podocytes and lower density, resulting in the glomerular basement membrane exposed, causing proteinuria.2. Mitochondrial targeting peptide SS-31 treatment prevented podocyte apoptosis induced by HOCl-alb, thus playing a protective role in renal function. Therefore, blocking the function of HOCl-alb and delaying the damage of mitochondria may become a new method for the treatment of diabetic nephropathy. |
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