Study On Kidney Injury And Energy Metabolism Characteristics Of Insulin Resistant Mice Induced By High Fat Feeding

With the continuous improvement of living standards,long-term high fat feeding leads to the increase of pet obesity,and also causes a variety of metabolic diseases in pets.Insulin is a key link in regulating energy metabolism.Long-term intake of high-energy food will lead to Insulin resistance(IR),and eventually lead to diabetes.Diabetic nephropathy is one of the common and serious complications of diabetes,but in fact,during hyperinsulinemia,the structure of glomeruli and renal tubules has undergone pathological changes.At present,most studies focus on the changes of kidney injury in the period of diabetes,and no systematic studies have been conducted on the changes of kidney injury before and after IR.At present,the cause of animal obesity is long-term intake of high-calorie food.Therefore,we established an animal model of IR by feeding high-fat diets,and observed the changes of kidney injury before and after IR from multiple perspectives and levels,such as kidney biochemical indexes,histopathology and glucose reabsorption capacity,so as to make a comprehensive judgment on the kidney injury.In addition,although previous studies on diabetic nephropathy have focused on the role of mitochondrial dysfunction and hypoxia in kidney injury,it is necessary to further clarify whether the energy metabolism mode of the kidney changes during IR,as well as the changes in the overall metabolic law and characteristics.Therefore,this study aims to establish the IR model of C57BL6 mice,and explore the relationship between kidney injury and energy metabolism during the process of IR.In this study,obesity and IR were induced in C57BL6 mice by feeding high fat diet.Glucose tolerance test and insulin resistance index were used to evaluate the occurrence of insulin resistance in low fat diet group(LFD)and high fat diet group(HFD).Serum and urine analyses were performed to assess changes in kidney function.HE,MASSON and PAS staining were used to observe the status of kidney tissue for pathological evaluation.The status of kidney podocytes and mitochondria were observed by transmission electron microscopy.Western blot was used to detect the expression of related proteins in the kidney,including insulin signaling pathway proteins,mitochondria-related proteins,autophagy proteins,inflammatory proteins,fibrotic proteins,aging proteins and apoptosis proteins.Renal glucose reabsorption related proteins were evaluated by immunohistochemistry.The key proteins LC3 and LAMP2 during renal autophagy were evaluated by immunofluorescence.The changes of renal ATP content,mitochondrial complex and lactate dehydrogenase activity were detected by the kit to comprehensively evaluate renal energy status.The results showed as follows: at the 6th week,the accumulation of glycogen and collagen fibers in renal tubules was observed in PAS and MASSON staining of HFD group.In HFD group,partial fusion occurred in the foot process of renal podocytes.The expression levels of inflammation-related proteins(IL-6 and TNF-α)in HFD group were significantly increased(P<0.001),and increased with the increase of time.The autophagy related proteins(Beclin1,LC3 and LAMP2)in HFD group were significantly lower than those in LFD group(P<0.001),but the expressions of senescence and apoptosis related proteins were significantly increased.Mitochondrial biogenesis related proteins(AMPK,PGC-1α,SIRT1/3)were significantly inhibited(P< 0.001),and the degree of inhibition increased with the increase of time.The mitochondrial fission protein(DRP1)in HFD group was significantly increased and increased with the increase of time(P < 0.001).More mitochondria in the state of division were observed by electron microscopy,and the mitochondrial fusion protein(OPA1,MFN2)was also significantly higher than that in LFD group(P<0.001).ATP in kidney was significantly higher than that in LFD group.Increased activity of fructose phosphokinase(P<0.05).At the 8th week,HFD group developed insulin resistance.The accumulation of kidney glycogen and collagen fiber increased in HFD group.HFD group increased the fusion of the foot process of renal podocyte,the basement membrane was uneven,and part of the basement membrane was thickened.Autophagy related proteins in HFD group were significantly lower than those in LFD group(P < 0.001).Mitochondrial fusion proteins(MFN2 and OPA1)in HFD group were significantly higher than those in LFD group(P < 0.05).The mitochondria of HFD group were spherical and began to appear swollen and mitochondria with unclear crista structure.In addition,the activities of fructose phosphokinase and lactate dehydrogenase were significantly increased compared with the LFD group(P<0.05).ATP content in kidney increased further and was significantly higher than that in LFD group.At week 10,renal insulin pathway was inhibited in HFD mice.There were significant changes in kidney injury related indexes(Scr and BUN)in HFD group(P<0.05).The accumulation of kidney glycogen and collagen fiber increased in HFD group.In HFD group,a large number of renal podocytes fused with the foot process and the basement membrane thickened.The expression levels of mitochondrial fusion proteins(MFN2 and OPA1)in HFD group were significantly lower than those in LFD group(P < 0.001).Through electron microscopy,the swelling and mitochondrial increase in HFD group were observed.The activities of fructose phosphokinase and lactate dehydrogenase were further enhanced.ATP content in kidney decreased significantly compared with that in the 8 weeks,but was still higher than that in the LFD group.At the 12 th week,the glomeruli in HFD group were swollen and matrix deposition was obvious,the tubular lumen of renal tubules was narrow,epithelial cells were shed,the glycogen deposition of renal tubules was reduced,and a large number of lipid vesicles appeared in renal tubules.In HFD group,a large number of renal podocytes fused with podocytes,and some podocytes shed.Kidney autophagy,mitochondrial biogenesis and fusion related proteins were severely inhibited(P <0.001).Through transmission electron microscopy,the mitochondria of renal tubules were fine,the ridge structure was fuzzy,and a large number of vacuolar mitochondria appeared.The activities of fructose phosphokinase and lactate dehydrogenase were further enhanced,but ATP content was significantly decreased compared with that at 10 weeks,but it was still not lower than that in LFD group(P<0.001).The results showed that high fat diet could induce IR in C57BL6 mice.After the development of IR,glomerular matrix deposition increases,inflammatory response and apoptotic pathway are enhanced,serum creatinine and other indicators are changed,kidney dysfunction occurs,at the same time,kidney cell autophagy decreases and cell senescence increases.Prior to renal IR,mitochondrial biogenesis is reduced,but it can still provide significant energy to the kidney through enhanced mitochondrial fusion and through anaerobic glycolysis;After the occurrence of IR in the kidney,mitochondrial fusion is reduced,mitochondrial production of ATP is reduced,and anaerobic glycolysis of the kidney is further enhanced.