| IntroductionThe lymphatic circulation forms an accessory pathway to return excess fluid, lymph cells, electrolytes and proteins from the renal tissues spaces back to the blood stream and thus plays an important role in maintaining fluid stability. We have studied the lymphatic circulation for more than300years, but the progress of the research is still far behind other systems. We have made clear the distributions of lymphatic vessels and lymph nodes, established the system of lymphatic circulation. But the understanding of the occurrence, formation, function and regulation of lymphatic vessels is far not enough. Unlike the blood circulation and neural system, lymphatic circulation is not restored as lymph vessels are not connected in kidney transplantation surgery. Lymph circulation can also be destroyed in inflammation, cancer, trauma and surgical procedure. The limited understanding of the lymphatic circulation blocked the development of medicine. Now the lymphatic circulation research has gradually started. There have been some reports about the lymphatic circulation disorders in heart, brain, liver and gastrointestinal organ but little has been done in kidney.Early papers demonstrated that acute ligation of hilar lymphatic ducts has significant effect on renal function and ligation of the thoracic duct induced diffuse sclerosis in renal parenchyma. However, our knowledge of renal lymphatic is very limited and their importance is almost always neglected in kidney transplant surgery practice. Our earlier studies found that the rats exhibited renal dysfunction and proteinuria after renal lymphatic ligation, but its effect and mechanism has not been closely studied.The progression of chronic renal failure is characterized by the loss of renal cells and their replacement by extracellular matrix and fibroblasts. In recent years apoptosis has been implicated in the progressive loss of renal cells. Apoptosis is a programmed cell death, including the update of the body’s normal cells and the depletion of abnormal cells. Abnormal apoptosis in pathological state is closely related to the development of many diseases. Apoptosis is a complex process that proceeds through two main pathways (extrinsic and intrinsic). Intrinsic pathway is also known as mitochondrial/cytochrome C (Cytc) pathway. Mitochondrial is the control center of the cell life, it is not only the center of the respiratory chain and oxidative phosphorylation, but also the center in regulation of apoptosis. The intrinsic pathway is triggered by mitochondria in response to intracellular injuries such as oxidative stress. The delicate ratio between proapoptotic and antiapoptotic proteins of the Bcl-2family increases and the permeability of the outer mitochondrial membrane increases. CytC is released into the cytoplasm and the downstream apoptotic pathways are activated. Eventually caspase3is activated to induce apoptosis. During the extrinsic apoptotic pathway, the tumor necrosis factor (TNF) family ligand such as Fas ligand (FasL) binds to its receptor and activates the extrinsic pathway initiator caspase, caspase-8(casp8). Casp8is activated and initiates apoptosis by direct cleavage of the downstream common effector caspases such as caspase3. We have confirmed the apoptosis of renal tubular epithelial cells in rats after ligation of renal lymphatic vessels. We wonder what are the roles of the two apoptotic pathways in this process? And what is the mechanism of apoptosis?To elucidate the association of renal lymphatic ligation with the progression of renal disease, renal cell apoptosis and its regulating genes and proteins, we studied both the extrinsic and intrinsic pathways of apoptosis in mononephrectomized rats with renal lymph circulation disorder.Aims1. To build the renal lymph circulation disorder model;2. To observe the effect of renal lymph circulation disorder in rats;3. To find the mechanisms of renal tubular epithelial cell apoptosis caused by renal lymphatic circulation disorder.Materials and Methods1. Animal experimentMale Sprague-Dawley rats, aged12weeks and weighing250-300g, were adaptive fed for7days. A total of54rats were divided randomly into3groups:rats with left renal lymph ligation and right nephrectomy (KL group, n=18), rats with only right nephrectomy (KN group, n=18) and sham-operated rats with two non-ligated kidneys (sham group, n=18). Renal lymphatic ligation was performed as below:the renal hilum trunks were ligated with fine silk sutures (8-0) microscopically (X12.5), and the fibroadipose tissue at the upper and lower poles of the kidneys was ligated to occlude capsular lymphatics. Sham-operated rats were used as controls. Rats were killed in groups (6in each group) at daysl,7and14after surgery, and the kidneys were removed. Creatinine concentration and24-hour urinary protein were measured.2. Hematoxylin and eosin (HE) stainingKidney specimen was fixed in4%paraformalde-hyde and embedded in paraf fin, and4u m sections were cut. Sections were subjected to routine staining with hematoxylin and eosin and viewed by a Nikon e90i microscope.3. Detection of apoptosis Apoptosis was determined using the terminal deoxynucleotidyl transferase dUTP nick-end-labeling (TUNEL) kit according to the manufacturer’s instructions. Slides were analyzed under a Nikon e90i microscope. The number of apoptotic cells in10non-overlapping fields on each slide was counted by a research staff blinded to the slide identity.4. Assessment of renal oxidative stress and superoxide dismutase activityRenal ROS production was measured by an enzyme-linked immunosorbent assay (ELISA) kit. Absorbance was determined with an ELISA reader at450nm against620nm as reference. MDA level was detected by the thiobarbituricacid method. Absorbance was measured at532nm by spectrometry. SOD activity was measured by the xanthine oxidase method. Absorbance was determined at550nm by spectrometry. All protein concentrations of renal tissue homogenate samples were determined with the Bradford method.5. Real-Time RT-PCRTotal RNA was extracted from whole kidney using TRIzol reagent. mRNA expression of Bax, Bcl-2and FasL were determined by real-time polymerase chain reaction (PCR) with the ABI Prism7500sequence detection system. GAPDH was used as the housekeeping gene.6. Western blotTotal tissue protein was extracted for testing Bax, Bcl-2and FasL, while cytoplasmic protein was extracted for testing cytochrome c (cyt-c)7. Determination of activities of caspase3, caspase8and caspase9The activities of caspase3, caspase8and caspase9were determined by using the corresponding activity detection kits following the manufacturer’s instructions. 8. Statistical analysisData were presented as mean±SE and was analyzed by one-way analysis of variance (ANOVA) and Student’s t test using SPSS software package (SPSS17.0). P<0.05was considered statistically significant.Results1. Renal functionCompared with the sham group, the levels of urine protein and serum creatinine in both KN and KL groups were significantly increased on day7(P<0.05). On day14, the levels of urine protein and serum creatinine in KN group were reduced to levels that were not significantly different from these in sham group; whereas these parameters were further increased in KL group (P<0.01)2. HE stainingHistological analysis was performed after HE staining. As shown in Fig. C, epithelial cell degeneration, detachment and atrophy were obvious in KL group; whereas all the above changes were less obvious In KN group. In sham group, the above changes were not found.3. Detection of apoptosisIn sham group, apoptotic cells were sparse at each time point. In KL group, apoptotic cells were significantly increased on dayl (P<0.05) and peaked on day14(P<0.01).In KN group, apoptotic cells were significantly increased on day7(P<0.05), but did not further increase on dayl4(P<0.05). Compared with KL group, apoptotic cells were significantly less in KN group at each time point (P<0.05)4. Measurements of ROS, MDA and SODCompared with the sham group, ROS production was significantly increased from day7and remained at a high level (P<0.05);kidney MDA level was increased (P<0.05) while the SOD activity was decreased (P<0.05) in KL group. In contrast, all the above parameters were not significantly different between KN group and sham group at each time point.5. Expression of Bax, Bcl-2, FasL genesAfter operation, Bax and FasL mRNA levels in KL group increased on dayl (P<0.05) and peaked on day14(P<0.01). While Bcl-2mRNA level decreased in KL group from dayl (P<0.05) and remained lower than controls(P<0.01). There were no significant changes in Bax and Bcl-2mRNA expressions in KN group compared to the sham. The mRNA expression of FasL increased from day7(P<0.05) in KN group compared to the sham, but still lower than the KL group (P<0.05)6. Tissue levels of Bax, Bcl-2, cytc, FasL proteinsThe levels of Bax, cyt-c, FasL proteins in ligated kidneys were increased (P<0.05) while the levels of Bcl-2protein in ligated kidneys were significantly decreased (P<0.05)from dayl compared with the sham group. The FasL protein level increased from day7compared with the sham group (P<0.05), but still lower than in the KL group (P<0.05). There were no significant changes in the levels of Bax, cyt-c and Bcl-2in KN group compared with the sham group.7. Activities of caspase3, caspase8and caspase9The activities of caspase3, caspase8and caspase9were increased from day1and maintained at a high level in KL group compared with the sham group (P<0.05). The activity of caspase8increased from day7in KN group compared to the sham (P<0.05), but lower than KL group (P<0.05). There were no significant differences of the activities of caspase3and caspase9in KN group compared with sham groupConclusion1. We confirmed that renal lymph circulation disturbance led to renal dysfunction and renal cell apoptosis. The apoptosis of renal cells increased after ligation of renal lymph vessels, mainly distributed in the dilated or atrophied tubules.2. Our results showed for the first time that both the intrinsic and extrinsic pathways participated in apoptosis in mononephrectomized rats with renal lymph circulation disorder. The mechanism might relate to the oxidative stress caused by lymph vessels ligation, suggestive of an essential role of renal lymphatic circulation in the maintenance of tubular integrity and function. IntroductionIn recent years, the incidence of chronic kidney diseases was significantly increased. The incidence rate of end stage renal disease (ESRD) increased from39.4/million to98.3/million per year from1988to2002. The rate will continue to increase by4.1%per year in the next10years. In China, the newly entering dialysis patients accounted for41.7%of the total number of dialysis patients in1999and maintained a higher trend. The third National Health and Nutrition Examination Survey of the America showed that, ESRD only accounted for0.6%in the total of patients with CKD, which meant more patients with relatively earlier stage. We can’t find the exact causes of many chronic renal impairments, which lead to the lack of effective treatment. Thus, it is urgent to find the risk factors for renal damage and to take early interventions.Unlike the blood circulation and neural system, lymphatic circulation is not restored as lymph vessels are not connected in kidney-transplantation surgery. Lymph circulation can also be destroyed in inflammation, cancer, trauma and surgical procedure. There have been some reports about the lymphatic circulation disorders in brain, liver and gastrointestinal organs but little has been done in kidneyKidney transplant is an effective treatment of ESRD, but it is faced with two major problems:kidney shortages and the loss of renal function in late transplant stage. In renal transplant patients, the long-term survival situation is not optimistic. Chronic allograft nephropathy (CAN) remains to be the main reason for late renal allograft dysfunction. CAN is divided to3grades according to their severity. The specific reasons for CAN are still not clear. According to present findings, many immune or (and) non-immune factors can lead to the occurrence of CAN. Immunosuppressants have been widely used in the clinical application to prevent rejection, but still couldn’t prevent CAN. There are no reports about the effective treatment of CAN by any immunosuppressant. Therefore, it is very significant to study of the non-immune factors.Our earlier study found that the rats exhibited renal interstitial fibrosis and tubular atrophy after renal lymph ligation. According to the "Banff97" standard, interstitial fibrosis and tubular atrophy are the main indicators of CAN. Our early experiments also showed high expression of TGF-β1in renal tubules and interstitium. High expression of TGF-β1and other cytokines play an important role in the development of CAN. Thus we wondered whether renal lymph circulation disorder could be one of the factors causing CAN.Renal interstitial fibrosis is a common feature of all chronic kidney diseases. It is the link between the progressive loss of renal function and kidney diseases. Renal fibrosis has been associated with the activation of interstitial fibroblasts to give rise to collagen secreting myofibroblasts. However, different studies have shown that in addition, myofibroblasts can also originate from renal tubular epithelial and endothelial cells that undergo EMT. EMT is a pathological process of renal interstitial fibrosis. It is a transdifferentiation variant and is also recognized as the mechanism for the formation of fibroblasts in damaged tissue. In this process, epithelial cells transdifferentiated into the matrix remodeling cells. These epithelial cells lose their epithelial polarity, cell adhesion molecules, cell cell and cell-matrix connection. This process is also accompanied by the gain of the mesenchymal cell markers (such as vimentin, fibronectin protein, smooth muscle actin and N-cadherin protein), and the loss of epithelial markers (such as E-cadherin protein). TGF-β1is the most important factors that cause EMT and BMP-7can effectively suppress TGF-β1-induced EMT. Though interstitial fibrosis and EMT have been widely studied, whether renal lymphatic circulation disorder can cause EMT and the role of TGF-β1are still unclear. Combined with our previous findings, we assumed that the EMT was involved in the pathological process and did further researches.In addition, studies have shown that angiotensin Ⅱ (AngⅡ) exists in renal lymphatic vessels. AngⅡ plays a certain role for EMT generation and is a major driving force for the occurrence of EMT. Whether AngⅡ increased after renal lymphatic ligation? What is the role of AngⅡ in EMT in this experiment?To solve these problems, we designed the following experiment.Aims1. To observe whether renal lymph circulation disorder can induce renal fibrosis or not2. To explore whether EMT participates in renal fibrosis induced by the renal lymph circulation disorder;3. To find the mechanisms of renal fibrosis induced by renal lymph circulation disorder.Materials and Methods1. Animal experimentStudies were performed in male SD rats (250-300g). A total of36rats were divided randomly into2groups:rats with left renal lymph ligation and right nephrectomy (KL group,n=18), rats with only right nephrectomy (KN group, n=18). Renal lymphatic ligation was performed as below:the renal hilum trunks were ligated with fine silk sutures microscopically, and the fibroadipose tissue at the upper and lower poles of the kidneys was ligated to occlude capsular lymphatics. Rats were killed in groups (6in each group) at days14,28and56after surgery, and the kidneys were removed. Creatinine concentration and24-hour urinary protein were measured.3. Masson and PAS stainKidney tissues were fixed in4%paraformaldehyde.4nμm thick slices were made after conventional dehydration and paraffin embedding. The Masson stain were carried out by dewaxing, hematoxylin staining, spring red acidic fuchsin solution staining, aniline blue liquid staining, dehydration. The PAS stain were carried out by dewaxing, periodic acid solution staining, Schiff dye staining, hematoxylin staining.3. Transmission Electron MicroscopyKidney tissue was taken from corticomedullary junction. Tissues were thinly sliced, placed immediately in primary transmission electron microscopy (TEM) fixative, and prepared as previously described. A transmission electron microscope was used to view all samples.4. Assessment of renal oxidative stressRenal ROS production was measured by an enzyme-linked immunosorbent assay (ELISA) kit. Absorbance was determined with an ELISA reader at450nm against620nm as reference. MDA level was detected by the thiobarbituricacid method. Absorbance was measured at532nm by spectrometry. SOD activity was measured by the xanthine oxidase method. Absorbance was determined at550nm by spectrometry. All protein concentrations of renal tissue homogenate samples were determined with the Bradford method.5. Real-Time RT-PCRTotal RNA was extracted from whole kidney using TRIzol reagent. mRNA expression of FSP1, BMP7, a SMA, E-cadherin and Gapdh were determined by real-time polymerase chain reaction (PCR) with the ABI Prism7500 sequence detection system. GAPDH was used as the housekeeping gene.6. Western blotTotal tissue protein was extracted for testing FSP1, BMP7, a SMA, E-cadherin.7. Determination of AngⅡ、AT1R、renin and activated reninAngⅡ、ATlR、renin and activated renin were determined by using the corresponding Elisa kits following the manufacturer’s instructions.8.Determination of TGFβ1、p-smad2/3TGF β1、p-smad2/3were determined by using the corresponding Elisa kits following the manufacturer’s instructions.9. Statistical analysisData were presented as mean±SE and was analyzed by one-way analysis of variance (ANOVA) using SPSS software package (SPSS17.0). P<0.05was considered statistically significant.Results1. Renal functionProteinuria and serum creatinine in KL was significantly raised compared to the KN group during the course of the experiment.2. Masson and PAS stainMasson staining showed that there are local brush border loss of renal tubular epithelial cells, vacuolar degeneration, cavity expand, cell disintegration shedding; PAS staining showed that there are local brush border loss of renal tubular epithelial cells, vacuolar degeneration, cavity expand, cell disintegration shedding, bare basement membrane formation. While in KN group.similar changes are not obvious.3. Transmission Electron Microscopy The KN kidneys showed well-preserved tubular epithelial cells exhibiting abundant mitochondria and surrounding thin basement membrane, whereas the ligated kidneys showed irregularly wrinkled basement membranes and swelling of the epithelial cells. The intertubular area of the KL kidneys was expanded in a fibrotic matrix with fibroblast-like cells, and the capillaries were collapsed.4. Renal oxidative stressThe ROS and MDA increased in KL group while SOD decreased compared with KN group. This meant that oxidative stress occurred by renal lymph ligation in KL group.5. RT-PCRThe mRNA level of FSP1, a SMA increased while BMP7and E-cadherin decreased in KL group compared with the KN group. FSP1, a SMA and E-cadherin are EMT markers and their changes prove the occurrence of EMT.6. Western blotThe protein level of FSP1, a SMA increased while BMP7and E-cadherin decreased in KL group compared with the KN group. FSP1, a SMA and E-cadherin are EMT markers and their changes prove the occurrence of EMT.7. Determination of AngⅡ、AT1R、renin and activated reninThe Ang Ⅱ level of the KL group increased compared with the KN group while there were no significant changes in AT1R, renin and activated renin in KL group. This reminded us that the renin-angiotensin system was not activated and did not participate the renal fibrosis process induced by renal lymphatic circulation disorder.8. Determination of TGF β1、 p-smad2/3The levels of TGF β1、p-smad2/3were all increased in KL group compared to the KN group. This reminded us that TGF β1-smad pathway participated the renal fibrosis induced by renal lymphatic circulation disorder. Conclusion1. We confirmed that the renal lymphatic circulation disorders could lead to renal dysfunction and renal fibrosis;2. EMT is one way by which renal lymph circulation disorder can cause renal fibrosis;3. We discussed the mechanisms of renal fibrosis caused by renal lymphatic circulation disorder. TGF β1-smad pathways was involved in fibrosis and EMT, while the RAS did not participate in this process. IntroductionThe incidence of chronic kidney disease (CKD) increased in recent years and it is gaining increasing medical and economical importance. The incidence of chronic kidney disease in China is about11%, and about1%of the patients will develop end-stage renal failure (ESRD). If the cost of each patient on dialysis is about RMB100,000, our country will pay several billion on it. Plus the other cost of the treatment of this disease and its complications, the total number is amazing. And the cost of medical resources is also enormous. So it is very important to prevent and treat CKD. However there is not enough reliable information about disease course, treatmentvariation, and the impact of treatments under routine clinical conditions on patient survival, quality of life, and costs.Silent information regulator factor2(sir2) is a histone deacetylase found in yeast cells, which is dependent on nicotine adenine dinucleotide (NAD). Sir2homologous protein family sirt has seven sirt analogs in mammalian:silent information regulator relating enzyme1-7(sirtl-sirt7). Each protein has its own targets and cellular localization. Sirt family proteins are highly conserved in evolution and are widely distributed in prokaryotes and eukaryotes. They play their important role through NAD-dependent enzyme catalytic activity in the cellular stress responses, such as double-stranded DNA damage, cell cycle and apoptosls.In the past five years, sirt3became a hot spot for the researchers. More and more scholars observed sirt3as a mitochondrial sirtuin that can regulate the metabolism and oxidative stress. Sirt3is a NAD+dependent deacetylase with the desacetyl activity. It is located on chromosome11and the gene length is21kb. Sirt3is highly expressed in muscle, liver, heart, kidney, and brown adipose tissue. The long term sirt3exists in mitochondria, cytoplasm and nucleus, while the short term sirt3exists only in mitochondria. Mitochondrial sirt3plays an important role in cellular energy metabolism, oxidative stress and apoptosis. Mitochondria are the main source of reactive oxygen species (ROS), ROS production increases when cells are subjected to stresses. SOD is the main defense factor against free radicals in the mitochondria. SOD can inhibit free radical chain reaction by disproportionation reaction and maintain the balance of free radicals in the body. When cells are damaged, the damage in the cells accumulates and leads to respiratory electron transport chain disruption, oxidative phosphorylation disorders, ATP synthesis decreasing and increased permeability of the mitochondrial membrane directly or indirectly. Respiratory electron transport chain disruption will further increase the production of ROS, thus forming a vicious cycle. When the body can not compensate this vicious cycle, apoptosis occurs. Over expression of sirt3can enhance respiration, reduce the generation of oxygen free radicals, reduce membrane potential, promote cyclic adenosine monophosphate response element binding protein phosphorylation, reduce ROS production, thereby reduce the oxidative stress-induced apoptosis. Meanwhile, it can also increase cell repair. Other studies have shown that the accumulation of ROS can cause cardiac hypertrophy and sirt3can prevent the occurrence of cardiac hypertrophy by the Foxo3a-dependent antioxidant mechanism. In addition, studies have shown that sirt3deficient mice can develop cardiac hypertrophy and interstitial fibrosis after8weeks though their outward keeps normal. Overreaction will happen when use the cardiac hypertrophy agonists, while sirt3transgenic overexpression mice can prevent this overreaction. We can see that sirt3plays a protective effect in tissues by reducing the level of intracellular ROS and interventing oxidative stress. It may become a new target. There are limited reports about the protective effect of sirt3on tissues. Most of the researches are focused on adipose tissue, cardiac muscle and nervous system. There are rare reports about kidney, even less in animal gene intervention.Our previous study confirmed that the renal lymphatic circulation disorders could lead to rat kidney dysfunction and oxidative stress played an important role in this process. So it is a suitable animal model to study sirt3. Sirt3can reduce the level of intracellular ROS and plays a protective role in aging-related diseases such as cardiovascular, cerebrovascular diseases and neurodegenerative diseases. It will be a new molecular target if we find out its role in the kidney, which will play an important role in the prevention and treatment of kidney disease.Aim1. To find out the role of sirt3in kidney and thus find a new target for the prevention and treatment of kidney diseases.2. To discuss the mechanisms of the effect of sirt3on kidney.Materials and Methods1. Animal experimentMale Sprague-Dawley rats, aged12weeks and weighing250-300g, were adaptive fed for7days. A total of72rats were divided randomly into4groups:rats with left renal lymph ligation and right nephrectomy (KL group, n=18), rats with left renal lymph ligation and right nephrectomy and also Lenti-sirt3injection (Lenti group, n=18);rats with left renal lymph ligation and right nephrectomy and GFP virus injection (GFP group, n=18); sham-operated rats (sham group, n=18). Renal lymphatic ligation was performed as below:the renal hilum trunks were ligated with fine silk sutures (8-0) microscopically (X12.5), and the fibroadipose tissue at the upper and lower poles of the kidneys was ligated to occlude capsular lymphatics.5X10’TU Lenti-sirt3/GFP virus was injected through tail vein per rat at day7after surgery. Sham-operated rats were used as controls. Rats were killed in groups (6in each group) at days7,14and28after surgery, and the kidneys were removed. Creatinine concentration and24-hour urinary protein were measured.2. Assessment of renal oxidative stress and superoxide dismutase activityRenal ROS production was measured by an enzyme-linked immunosorbent assay (ELISA) kit. Absorbance was determined with an ELISA reader at450nm against620nm as reference. MDA level was detected by the thiobarbituricacid method. Absorbance was measured at532nm by spectrometry. SOD activity was measured by the xanthine oxidase method. Absorbance was determined at550nm by spectrometry. All protein concentrations of renal tissue homogenate samples were determined with the Bradford method.3. Real-Time RT-PCRTotal RNA was extracted from whole kidney using TRIzol reagent. mRNA expression of caspase3、collagen I、sirt3and Gapdh were determined by real-time polymerase chain reaction (PCR) with the ABI Prism7500sequence detection system.GAPDH was used as the housekeeping gene.4. Western blotTotal tissue protein was extracted for testing caspase3, collagen I and sirt3.5. ImmunohistochemistryParaffin sections were incubated with antisera against caspase3, collagen I and Sirt3, using a standard avidin-biotin peroxide technique together with heat-mediated antigen retrieval. The negative control included replacement of the primary antibodies with phosphate-buffered saline (PBS), which showed no staining. Photomicrographs were taken using under microscope6. Statistical analysisData were presented as mean±SE and was analyzed by one-way analysis of variance (ANOVA) and Student’s t test using SPSS software package (SPSS17.0). P<0.05was considered statistically significant.Results1. Clinical parametersCompared to the sham group, proteinuria and blood creatinine in KL rats were significantly raised from day7and reached a peak at day28. These parameters were improved in Lenti group at day14and day28compared to the KL group, but were still high compared to the sham group. There were no significant changes between KL group and GFP group.2. Oxidative stressCompared to the sham group, ROS and MDA levels in KL rats, GFP rats and Lenti rats were significantly raised from day7and reached a peak at day28. These parameters decreased in Lenti group from day14compared to the KL group. Compared to the sham group, SOD levels in KL rats, GFP rats and Lenti rats were significantly decreased from day7. This parameter increased in Lenti group from dayl4compared to the KL group. There were no significant changes between KL group and GFP group.3. RT-PCRCompared to the sham group, caspase3and collagen I levels in KL rats, GFP rats and Lenti rats were significantly raised from day7and reached a peak at day28. There were no significant changes between KL group and GFP group. Sirt3levels of KL group decreased increasingly from day7compared with the sham group. Compared to the sham group, the level of sirt3decreased at day7, increased at dayl4and decreased again to a normal level at day28. Compared to the KL group, caspase3and collagen I levels in Lenti rats decreased from day14, while sirt3levels increased from day14and remained at a higher level. This meant that sirt3expression increased after injection of lenti virus and further decreased the expression levels of caspase3and collagen I.4. Western blotCompared to the sham group, caspase3and collagen I levels in both KL rats, GFP rats and Lenti rats were significantly raised from day7and reached a peak at day28. Sirt3levels of KL group decreased increasingly from day7compared with the sham group. There were no significant changes between KL group and GFP group. Compared to the sham group, the level of sirt3decreased at day?, increased at day14and remained at a high level. Compared to the KL group, caspase3and collagen I levels in Lenti rats decreased from day14, while sirt3levels increased from day14and remained at a higher level. The result is familar to the rt-pcr result.5. ImmunohistochemistryCaspase3was hardly observed in kidneys of the sham group. After ligation, caspase3staining was obviously seen in the renal tubules. The staining of caspase3decreased in Lenti group compared with the KL group and GFP group. In contrast, there was sirt3staining in the tubules of sham-operated rat kidneys. However, staining was rarely seen in those areas of the ligated kidneys. After injection of Lenti-virus, the expression of sirt3increased compared to the KL group and GFP group. Immunohistochemical stainings indicated that collagen I was barely expressed in sham group. In contrast, in KL group and GFP group, staining was prominent in the tubulointerstitium and glomerular mesangium. The intensity of immunostaining from Lenti group was reduced, but was still stronger than sham group.Conclusion 1. Sirt3plays a protective role in kidney.2. The protective role may relate to oxidative stress.3. This research just confirmed the the general role of sirt3in kidney and further researches should be done. The studies of renal lymph circulation disorder rats are limited and maybe we should use other familiar animal models to study the exact mechanisms. |
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