| Mitochondria is an important organlle containing DNA in cell, regulating the metabolism of cell. Mitochondria is indispensible for the survival of cell. The main function of mitochondria is as follows:(1) the majority of ATP required by the body is produced by mitochondria;(2) clearance of the redundant fatty acids, prevention of non-esterified fatty acid into the blood circulation;(3) keeping the homeostasis of calcium, regulating the cell apoptosis and death pathway. Mitochondria may produce the reactive oxygen species(ROS), under physiological conditions, ROS will be eliminated by anti-oxidant enzymes, which keeps the balance between the production of ROS and the capability of anti-oxidant. Oxidative stress will occur when ROS was over-produced or the capability of anti-oxidant declined. Mitochondrial-derived oxidative stress can cause the open of mitochondrial permeability transition pore of endothelial cells, which mediates the release of inflammatory factors. The occurrence of inflammatory reaction will damage the homeostasis of the body. Mitochondrial dysfunction-induced the decrease of ATP production, oxidative stress and inflammatory reaction will influence the onset and progress of many diseases. Currently, mitochondrial dysfunction was proved to be associated with neurodegeneration, diabetes, aging and so on. For example, mitochondrial dysfunction occurs at the early stage of aging without the histological changes. Mitochondrial dysfunction is closely associated with renal diseases. The kidney tissues are rich in mitochondria, the damage of oxidative phosphorylation will cause mitochondrial dysfunction. In 2000, Doleris LM reported that 4 mitochondrial cytopathy patients were complicated with focal segmental glomerulosclerosis(FSGS), progressing to chronci renal failure(CRF).Another report indicated that mutated mt DNA accumulated in more than 80% of the kidney of FSGS, glomerular TFAM m RNA level increased significantly at the early stage of PAN, mt DNA number increased to 241%;whereas TFAM m RNA level decreased significantly, mt DNA number declined to 34% during the stage of FSGS. Our team also reported that inhibition of mitochondrial dysfunction can attenuate the proteinuria-induced chronic kidney disease in mice. These reports all indicated that mitochondrial dysfunction was closely associated with kidney diseases.CRF is the common endpoint of all kinds of renal diseases, which is characterized by the progressive nephron damage resulting in the disorder of excretory and endocrine system with poor prognosis. The therapy of CRF was mainly the protection of residual nephrons, slowing the progress. Hence, it is imperative to seek the new therapeutic target. Mitochondrial dysfunction-induced lower ATP production and increase of ROS and inflammatory factors was associated with the onset and progress of CRF, which promote CRF to end stage renal disease(ESRD). On the other hand, renal tubulointerstitial fibrosis is the common pathway of the development of CRF to ESRD. Renal tubulointerstitial fibrosis is closely associated with the severity of CRF.Therefore, we think that mitochondrial dysfunction is involved in the onset and progress of renal tubulointerstitial fibrosis of CRF, blocking mitochondrial dysfunction can alleviate renal tubulointerstitial fibrosis and associated symptoms of CRF.PPARs, ligand-activated nuclear receptor family, is the member of II type nuclear receptor superfamilies, including α、β、γ subtypes, among which PPARγ plays a major role in the normal kidney development 、 fat metabolism, regulating water-salt reabsorption、renal blood flow and activating RAAS. PPARγ can regulate anti-oxidant enzyme and resist oxidative stress, PPARγ activator plays a role in protecting the kidney and cell injury. Activating PPARγ inhibits the cannaboid I receptor expression induced by high glucose, and alleviate the inflammatory reaction and fibrosis ofmesangial cells; PPARγ activating can attenuate the renal interstitial fibrosis in UUO model. It is imperative to study the role of PPARγ activator in the kidney. Our previous study indicated that PPARγ activator can block mitochondrial dysfunction and attenuate aldosterone-induced podocyte injury and mesangial proliferation. In terms of the anti-oxidative stress and renal protective effects of PPARγ, we hypothesized that PPARγ activation can alleviate renal tubulointerstitial fibrosis of CRF through blocking mitochondrial dysfunction.Mitochondrial dysfunction usually associated with the inflammatory reaction, which is the adaptive reaction of body at the early stage. Continuous inflammatory reaction is likely to cause damage to the body. Compared with the specific immune reaction(T、B cell), natural immune is the first defense line against the microorganism attack, playing an important role in anti-infection immune. Natural immune can identify the non-infectious and infectious stimulus through identifying membrane and pattern recognization receptor. NLRP3 is one of the members if nucleotide binding oligomerization domain, can recruit the apoptosis-associated speck-like protein cantaining CARD and pro-caspase-1, which form NLRP3 inflammasome. NLRP3 inflammasome promotes the release of inflammatory factors, such as IL-18 and IL-1β. Mitochondrial dysfunction and CRF are usually associated with aggravated inflammatory reaction, the release of IL-18 and IL-1β induced by NLRP3 inflammasome is proved to be associated with renal diseases. Our previous study indicated that knockout of NLRP3 inflammasome gene can inhibit mitochondrial dysfunction and attenuate albumin-induced MPTC injury. NLRP3 inflammasome is involved in the onset and progress of UUO model. The typical pathological changes of UUO is renal tubulointerstitial fibrosis. Renal tubulointerstitial fibrosis is closely associated with the progress of CRF. Hence, we postulated that downregulation of NLRP3 inflammasome can attenuate renal tubulointerstitial fibrosis of CRF throughblocking mitochondrial dysfunction.It is absolutely possible to understand the role of mitochondrial dysfunction in CRF onset/progress, which provided new insight to the prevention/therapy of CRF. Meanwhile the in-depth study of PPARγ and NLRP3 inflammasome is helpful for seeking novel therapeutic target.Part I Mitochondrial dysfunction mediates renal tubulointerstitial fibrosis of chronic renal failureObjective: To explore the role of mitochondrial dysfunction in renal tubulointerstitial fibrosis of CRF.Methods: Mouse PTC were cultured in vitro, PTC were pretreated with Mn TBAP, and were divided into control group, TGF-β1 group, and Mn TBAP+TGF-β1 group. C57BL/6J mice were under 5/6 nephrectomy, the mice were injected Mn TBAP intraperitonealy. And the mice were divided into 4 groups: control group, 5/6 nephrectomy group, Mn TBAP group, and Mn TBAP+5/6 nephrectomy group. The expression of E-cadherin, Vimentin, α-SMA, NLRP3 and PPARγ were examined by real-time PCR and western blot. Mitochondrial ultramicrostructure was displayed by electromicrograph morphometry. ROS production was determined by DCFDA fluorescence. The mitochondrial membrane potential was determined by JC-1 staining. Mitochondrial DNA(mt DNA) copy number was detected by real-time PCR. Urine protein/urea ratio, serum urea nitrogen and creatinine and hemoglobulin was determined by ELISA method, systolic blood pressure of mice tail was measured by non-invasive blood pressure monitoring equipment, spleen was weighted, renal pathology was detected by massoon staining, the expression of renal cotex fibronectin、collagen-I、collagen-III were determined by real-time PCR and western blot.Results:(1) TGF-β1 decreased the expression of E-cadherin,PPARγ and increased the expression of Vimentin and α-SMA time-dependently;(2) TGF-β1 increased the production of ROS, decreased the mitochondrial membrane potential and mt DNA copy number. The mitochondrial ultramicrostructura morphology displayed vacuolization and decreased distribution in PTC;(3) In vivo, urine protein/crea ratio increased significantly 2 weeks after 5/6 nephrectomy, systolic blood pressure increased obviously 8 weeks after 5/6 nephrectomy, in 12 weeks after 5/6 nephrectomy, spleen weight 、 serum nitrogen ure and creatinine increased significantly, hemoglobulin decreased significantly, Masson staining indicated tubolointerstitail fibrosis, renal tissue PTC mitochondrial morphology vaculoization. The mt DNA copy number and PPARγ m RNA level decresed significantly, NLRP3 m RNA levels increased significantly.(4) Mn TBAP blocked TGF-β1-induced ROS production, decreased mitochondrial membrane potential and mt DNA copy number. Mn TBAP also improved mitochondrial ultramircostructure, upregulated the expression for E-cadherin and inhibited the expression of Vimentin、α-SMA.(5) In vivo, Mn TBAP inhibited the tubulointerstitial fibrosis and the expression of fibronectin、collagen-I、collagen-III. The levels of serum creatinine、nitrogen urea、spleen weight、systolic blood pressure and urine protein/crea ratio declined, hemoglobin and mt DNA copy number increased.Conclusions: Mitochondrial dysfunction is involved in renal tubulointerstitial fibrosis and symptoms of CRF, blocking mitochondrial dysfunction can attenuate renal tubulointerstitial fibrosis and symptoms of CRF.Part II PPARγ attenuates mitochondrial dysfunction and renal tubulointerstitial fibrosis of chronic renal failureObjective: To observe the role of PPARγ in mitochondrial dysfunction and renal tubulointerstitial fibrosis of CRF.Methods: Mouse PTC were cultured in vitro, PTC were transfected transiently with pc DNA3-h PPARγ, and were divided into control group, vehi group, TGF-β1 group and PPARγ+TGF-β1 group. C57BL/6J mice were under 5/6 nephrectomy. Rosiglitazone was injected subperitoneally. The mice were divided into 4 groups:control group, 5/6 nephrectomy group, Rosiglitazone group, and Rosiglitazone +5/6 nephrectomy group. The expression of E-cadherin, Vimentin and α-SMA were examined by real-time PCR and western blot. Mitochondrial ultramicrostructure was displayed by electromicrograph morphometry. ROS production was determined by DCFDA fluorescence. The mitochondrial membrane potential was determined by JC-1 staining. mt DNA copy number was detected by real-time PCR. Urine protein/urea ratio,serum urea nitrogen and creatinine and Hb was determined by ELISA method, systolic blood pressure of mice tail was measured by non-invasive blood pressure monitoring equipment, spleen was weighted, renal pathology was detected by massoon staining, the expression of renal cotex fibronectin、collagen-I、collagen-III were determined by real-time PCR and western blot.Results:(1) PPARγ blocked TGF-β1-induced ROS production, decreased mitochondrial membrane potential and mt DNA copy number. PPARγ also improved mitochondrial ultramircostructure, upregulated the expression for E-cadherin and inhibited the expression of Vimentin、α-SMA.(2) In vivo, Rosiglitazone inhibited the tubulointerstitial fibrosis and the expression of fibronectin、collagen-I、collagen-III. The levels of serum creatinine、nitrogen urea、spleen weight、systolic blood pressure and urine protein/crea ratio declined, Hb and mt DNA copy number increased.Conclusions: PPARγ attenuate renal tubulointerstitial fibrosis and associated symptoms, which may be due to the inhibition of mitochondrial dysfunction.Part III The downregulation of NLRP3 attenuates mitochondrial dysfunction and renal tubulointerstitial fibrosis of chronic renal failureObjective: To observe the role of NLRP3 downregulation in mitochondrial dysfunction and renal tubulointerstitial fibrosis of CRF.Methods: Mouse PTC were cultured in vitro, PTC were transfected transiently with NLRP3 Si RNA, and were divided into control group, vehi group, TGF-β1 group, and NLRP3-/-+TGF-β1 group. The C57BL/6J mice were divided into 4 groups: control group, 5/6 nephrectomy group, NLRP3-/- group, and NLRP3-/- +5/6 nephrectomy group. The expression of E-cadherin, Vimentin and α-SMA were examined by real-time PCR and western blot. Mitochondrial ultramicrostructure was displayed by electromicrograph morphometry. ROS production was determined by DCFDA fluorescence. The mitochondrial membrane potential was determined by JC-1 staining. mt DNA copy number was detected by real-time PCR. Urine protein/urea ratio was determined by ELISA method, systolic blood pressure of mice tail was measured by optical plethysmography, serum urea nitrogen and creatinine were determined by biochemical equipment, spleen was weighted, Hb was measured by resistance method, renal pathology was detected by massoon staining, the expression of renal cotex fibronectin、collagen-I、collagen-III were determined by real-time PCR and western blot.Results:(1) The downregulation of NLRP3 blocked TGF-β1-induced ROS production, decreased mitochondrial membrane potential and mt DNA copy number. Thedownregulation of NLRP3 also improved mitochondrial ultramircostructure, upregulated the expression for E-cadherin and inhibited the expression of Vimentin、α-SMA.(2) In vivo, the downregulation of NLRP3 inhibited the tubulointerstitial fibrosis and the expression of fibronectin、collagen-I、collagen-III. The levels of serum creatinine、nitrogen urea、spleen weight、systolic blood pressure and urine protein/crea ratio declined, Hb and mt DNA copy number increased.Conclusions: The downregulation of NLRP3 attenuate renal tubulointerstitial fibrosis of CRF, which may be due to the inhibition of mitochondrial dysfunction. |
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