| BackgroundWith the continuous improvement of renal transplantation techniques and the advancement of immunosuppressive drugs,the short-term efficacy of renal transplantation has been remarkable,but the long-term survival rate has not been significantly improved.Nearly 50% of transplanted kidneys develop graft renal failure around 10 years after surgery and thus require resumption of hemodialysis or secondary transplantation therapy.In recent years,chronic graft renal failure due to non-immune renal fibrosis has attracted much attention and is a challenge to increase the long-term survival of renal transplantation.Renal tubules are the main bearers of renal reabsorption and secretion functions.Tubular epithelial cells are highly involved in the process of repair or fibrosis after renal injury as intrinsic cells of renal tubular mesenchyme.These cells mainly rely on fatty acid oxidation for energy.Impaired fatty acid oxidation in the kidney is a common feature in the developmental process of many diseases such as chronic kidney disease,cancer and fibrosis.Recent studies have shown that low levels of fatty acid oxidative capacity in renal tubular epithelial cells are associated with tubulointerstitial fibrosis.Increased glycolysis,on the other hand,leads to lactate and lipid accumulation,which in turn exacerbates acidosis,tissue damage and fibrosis.Accumulation of glycolytic intermediates in acute kidney injury and diabetic nephropathy leads to the production of toxic end products,which exacerbate fibrosis progression.Therefore,glucose-lipid metabolic homeostasis is critical for maintaining normal kidney function,but its relationship with transplant renal fibrosis is unclear.Whether targeting restoration of glucose-lipid metabolic homeostasis to delay the progression of transplanted renal fibrosis is feasible and the specific efficacy of this treatment also requires further study.Liver kinase B1(LKB1),a tumor suppressor,together with its most important downstream target,adenosine 5’-monophosphate(AMP)-activated protein kinase(AMPK),is the key regulatory hub for glucose and fatty acid metabolism,which is involved in the regulation of a variety of biological processes including cell polarity,cell cycle arrest,apoptosis,energy metabolism and hematopoietic stem cell maintenance.Glucose-lipid metabolism disorders caused by impairment of it’s signaling pathway may play an important role in the process of transplanted renal fibrosis,but the specific mechanism is still unclear.ObjectiveFirstly,to verify the existence of glucose-lipid metabolism disorders and the relationship between glycolysis and fatty acid oxidation regulated by LKB1-AMPK pathway and transplantation renal fibrosis;secondly,to further clarify the specific mechanism of LKB1-AMPK signaling pathway involved in transplantation renal fibrosis;and lastly,to explore the possibility that targeting LKB1-AMPK signaling pathway to restore the homeostasis of glucose-lipid metabolism can alleviate the transplantation renal fibrosis and provide a new treatment strategy and theoretical basis for delaying transplantation renal fibrosis and increasing the long-term survival of transplanted kidney with function.Finally,we will explore whether targeting the LKB1-AMPK signaling pathway to restore glucose-lipid metabolism homeostasis can reduce transplant kidney fibrosis and evaluate its efficacy in protecting renal function,so as to provide a new therapeutic strategy and theoretical basis for slowing down the transplant kidney fibrosis,and to provide new ideas for preventing and controlling the loss of function of the transplant kidney in clinical practice and for increasing the survival of the transplanted kidney in long-term.MethodsPart 1 Impaired LKB1-AMPK signaling pathway leads to disorders of glucolipid metabolism and promotes transplantation renal fibrosis1.Brown Norway rat kidney transplantation model was constructed and non-targeted metabolomics sequencing of transplanted kidney was performed to clarify the changes of sugar and lipid metabolism;2.Obtain serial sections of clinical transplant kidney puncture samples for immunohistochemical staining of Masson and LKB1,p-AMPK,and analyze the results of transplant kidney genome sequencing in conjunction with public databases to clarify whether the LKB1-AMPK pathway is associated with the degree of transplant kidney fibrosis;3.To construct stable transient cell lines of human renal proximal tubular epithelial cells HK-2 and rat renal tubular epithelial cells NRK with knockdown of LKB1,and to observe the alteration of fatty acid metabolism of the cells by Oil Red O and Nile Red staining,and the alteration of the glycolysis ability by detecting the extracellular acidification rate;4.To clarify the effects of knocking down LKB1 on collagen secretion and extracellular matrix(ECM)remodeling in HK-2 and NRK cells by western blot and RT-qPCR;5.The effects of knocking down LKB1 on epithelial mesenchymal transition(EMT)in HK-2 and NRK cells were clarified by cell scratch assay,Transwell cell migration assay,cytoskeletal staining of ghost pen cyclic peptide,as well as western blot and RT-qPCR;Part 2 Mechanism of impaired LKB1-AMPK signaling pathway leading to fibrosis in transplanted kidneys1.To clarify the correlation between the LKB1-AMPK-PPARα(perixisome proliferation-activated receptor alpha)axis and the degree of fibrosis in transplanted kidneys by western blot,and immunohistochemical staining results of Masson and PPARα in serial sections of clinical transplanted kidney puncture samples;2.To clarify whether the application of fenofibrate targeted activation of PPARα could reverse the fatty acid metabolism disorder caused by knockdown of LKB1 by oil red O,Nile Red staining,and cellular ATP content measurement;3.To determine whether fenofibrate-targeted activation of PPARα can reverse the increased collagen secretion,ECM remodeling and EMT process caused by impaired LKB1-AMPK-PPARα signaling pathway by western blot,RT-qPCR and cellular immunofluorescence;4.Serial sections of clinical transplant kidney puncture samples were subjected to immunohistochemical staining for Masson and LKB1 and ACOX1(acyl-Co A Oxidase 1),and analyzed in conjunction with the sequencing of the transplant kidney genome from public databases,and western blot results,to clarify whether the LKB1-AMPK-PPARα-ACOX1 pathway of the fatty acid peroxisomal βoxidation pathway is correlated with the degree of transplant kidney fibrosis;5.Constructed HK-2 stably transfected cell lines with knockdown of ACOX1 to clarify the correlation between ACOX1 and fibrosis;6.To determine whether overexpression of ACOX1 can reverse the increased collagen secretion,ECM remodeling,and EMT process caused by the impaired LKB1-AMPK-PPARα-ACOX1 signaling pathway by western blot,RT-qPCR,and cellular immunofluorescence;7.To clarify whether the LKB1-AMPK-HK1(hexokinase 1),which regulates glycolysis,is related to the transplantation kidney fibrosis by immunohistochemical staining of Masson and HK1 in RNA sequence,western blot,q RT-PC,and serial sections of clinical transplantation renal puncture samples from HK-2-shACOX1 cells,as well as by analyzing the results of transplantation renal genome sequencing from public databases.pathway regulating glycolysis is associated with the degree of fibrosis in transplanted kidneys;8.To determine whether inhibition of glycolysis by 2-DG can reverse the increased collagen secretion of renal tubular epithelial cells,the remodeling of ECM and the EMT process caused by the impairment of the LKB1-AMPK pathway by western blot,RT-qPCR and cellular immunofluorescence;Part 3 Drug-targeted restoration of glucose-lipid metabolic homeostasis inhibits transplantation renal fibrosis1.To determine whether the targeted activation of AMPK by A769662 can regulate the disorders of glucose and lipid metabolism in renal tubular epithelial cells by western blot,RT-qPCR,oil red O and Nile Red staining,extracellular acidification rate assay,and cellular ATP content assay;2.By western blot,RT-qPCR and cellular immunofluorescence techniques,it was determined whether the application of A769662 to target and activate AMPK to restore the balance of glucose and lipid metabolism could reverse the increase in collagen secretion,the remodeling of the ECM and the process of EMT in the renal tubular epithelial cells caused by the impairment of the LKB1-AMPK pathway;3.To clarify whether the application of metformin treatment could reduce the level of inflammatory response in transplanted kidneys by PAS staining,determination of TNF-α/IL-1β/MCP1 by ELISA,immunohistochemical staining and western blot;4.Construct the Brown Norway rat kidney transplantation model and apply fenofibrate,2-DG or metformin treatment to verify the above in vitro experimental results and the protective effect on renal function.ResultsPart 1 Impaired LKB1-AMPK signaling pathway leads to disorders of glucolipid metabolism and promotes transplantation renal fibrosis1.Decreased glycoconjugates and significantly increased lipid accumulation in the transplanted kidneys of Brown Norway rats confirmed the occurrence of disorders of glucose-lipid metabolism;2.Significant increase of fibrosis in LKB1 and p-AMPK staining-negative regions in clinical transplanted kidney samples,and sequencing results from public databases also showed that LKB1 and AMPK transcript levels were negatively correlated with the degree of fibrosis in transplanted kidneys(P<0.05),suggesting that there was an impaired LKB1-AMPK signaling pathway in the transplanted kidneys and correlated it with fibrosis;3.Lipid accumulation and extracellular acidification were significantly increased in HK-2-shLKB1 and NRK-shLKB1 cells,suggesting impaired fatty acid oxidation along with increased glycolysis;4.shLKB1 resulted in increased RNA and protein levels of genes related to type I and IV collagen secretion and ECM remodeling in both HK-2 and NRK cells;5.shLKB1 led to an increase in the migratory ability of HK-2 and NRK cells,a change in cell morphology from the typical cobblestone-like appearance of epithelial cells to a spindle-shaped appearance,and an increase in the RNA and protein levels of genes related to EMT,confirming the occurrence of EMT;Part 2 Mechanism of impaired LKB1-AMPK signaling pathway leading to fibrosis in transplanted kidneys1.The significant increase of fibrosis in PPARα-staining-negative areas in clinical transplanted kidney samples and the ability of shLKB1 to cause a decrease in PPARα expression confirmed the involvement of PPARα as a downstream of LKB1-AMPK in the fibrotic process;2.Fenofibrate-targeted activation of PPARα could reverse the decrease in lipid accumulation and ATP content in cells caused by shLKB1;3.Fenofibrate-targeted activation of PPARα was able to reverse the increased secretion of collagen types I and IV,ECM remodeling,and the EMT process caused by the impairment of the LKB1-AMPK-PPARα signaling pathway;4.The significant increase of fibrosis in ACOX1 staining-negative areas,the high co-localization of ACOX1 with LKB1,and the decrease of ACOX1 expression due to shLKB1 in the clinical transplanted kidneys confirmed the involvement of ACOX1 as a downstream of LKB1-AMPK-PPARα in the fibrotic process;5.shACOX1 resulted in increased collagen secretion,ECM remodeling,and EMT in renal tubular epithelial cells;6.Overexpression of ACOX1 was able to reverse the increased collagen secretion,ECM remodeling,and EMT processes resulting from the impaired LKB1-AMPK-PPARα-ACOX1 signaling pathway;7.The transcript levels of glycolysis-related genes were significantly increased after shACOX1,and2-DG treatment was able to significantly inhibit the RNA levels of genes related to collagen secretion;the public database analysis also showed that the transcript levels of genes for key enzymes of glycolysis were negatively correlated with the degree of transplantation renal fibrosis(P<0.05);and there was also a significant increase in the fibrosis of HK1-stained areas in the clinical transplantation renal puncture samples,these results suggest that fibrosis caused by impaired LKB1-AMPK pathway is associated with increased glycolysis;8.Inhibition of glycolysis by 2-DG reversed the increased secretion of collagen types I and IV by renal tubular epithelial cells,the remodeling of ECM and the process of EMT caused by the impairment of the LKB1-AMPK pathway;Part 3 Drug-targeted restoration of glucose-lipid metabolic homeostasis inhibits transplantation renal fibrosis1.Targeted activation of AMPK by A769662 could reduce lipid deposition,decrease the rate of extracellular acidification,and increase the ATP content of cells,which confirmed that it could reverse the disorders of glucose-lipid metabolism in renal tubular epithelial cells;2.Targeted activation of AMPK by A769662 restored the homeostasis of glucose-lipid metabolism and reversed the increased secretion of collagen types I and IV,ECM remodeling and EMT in renal tubular epithelial cells caused by the impairment of the LKB1-AMPK pathway;3.Metformin treatment was able to attenuate inflammatory cell infiltration,tissue TNF-α/IL-1β/MCP1/IL-6 levels,and reduce inflammatory responses in transplanted kidneys;4.In vivo experiments demonstrated that the application of fenofibrate,2-DG or metformin in Brown Norway rats receiving renal transplants could attenuate transplant renal fibrosis and protect renal function,with metformin having the most significant efficacy.Conclusion1.Glucose-lipid metabolism is disturbed in fibrotic transplanted kidneys and is associated with an impaired LKB1-AMPK signaling pathway in renal tubular epithelial cells;2.Impaired LKB1-AMPK signaling pathway leads to EMT in renal tubular epithelial cells which in turn increases type I and IV collagen secretion and ECM remodeling;3.Impaired β-oxidation of fatty acids in the peroxisomal pathway catalyzed by PPARα-ACOX1 and increased HK1-catalyzed glycolysis in transplanted kidneys lead to abnormalities in glucolipid metabolism;4.The application of fenofibrate,2-DG and metformin to target the restoration of glucose-lipid metabolism homeostasis can delay the transplantation of renal fibrosis to a certain extent,of which metformin also reduces the inflammatory response,which is a better efficacy for the protection of renal function.These results provide new perspectives for understanding the complex molecular mechanisms of transplant renal fibrosis,and new therapeutic strategies for delaying transplant renal fibrosis and increasing the length of time the transplanted kidney carries function. |
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