| BackgroundDiabetic kidney disease(DKD)occurs in type 1and type 2 diabetes mellitus and is the leading cause of end-stage renal disease worldwide.The current treatment options of DKD have remained limited and unchanged over the decades,at least partially because of the insufficient progress in deciphering the pathology and pathogenesis of DKD.More and more clinical and basic researches imply that there is a great relationship between DKD and lipid metabolism.There is growing evidence that abnormal lipid metabolism and renal lipid accumulation play a role in DKD pathogenesis [1-3].Overload of triglyceride(TG)and its metabolic product free fatty acid(FFA)in plasma can result in endothelial dysfunction and vascular damage by direct adhesion or inflammation [4].Excess extracellular FFA can also be imported into adipose and non-adipose cells and stored as TG in lipid droplets.Adipocytes have a unique capacity to store large amounts of excess TG in cytosolic lipid droplets.However,cells of non-adipose tissues,cardiomyocytes and podocytes for example,have a limited capacity for storage of lipids.Although TG is considered the most neutral and harmless type of intracellular lipid storage,the accumulation of TG in non-adipose cells can affect cell dysfunction and death by physical pressure.Alternatively,fatty acids can be converted to lipid intermediates like diacylglycerol,ceramides and fatty acyl-Co As.These lipid intermediates can impair cellular function,which is referred to as lipotoxicity [5-7] [plus1].In kidney,it leads to tubule interstitial inflammation and fibrosis in mild cases,and to renal failure and death in severe cases[8].Adipose triglyceride lipase(ATGL)is an important component of lipolytic process and the rate-limiting enzyme for the initiation of TG catabolism [9].Haemmerle,et al.[10] for the first time reported that there was a large amount of TG content in internal organs including cardiac muscle,liver and kidney in Atgl-/-mice compare to Atgl+/+ mice.Since then,many studies looked into the relationship between Atgl and cardiac muscle and liver [11-13].But there is little known about the relation between Atgl and kidney.Our pre-experiment reminded that there was a pronounced albuminuria in Atgl-/-mice,which suggested damage of glomerular filtration barrier.Our purpose of the research is to definitude the influence of Atgl gene deletion on kidney through observing the structure and function change of Atgl-/-mice,and to explore the possible mechanism by establishing ATGL down-regulated expression podocytes model.Materials and methods1.To determine the impact of Atgl deficiency on lipid metabolism in vivo,we assessed serum TG,FFA and total cholesterol(TC)concentration and TG concentration in cardiomyocyte,liver and kidney of Atgl-/-mice.2.Albumin /creatinine ratio(ACR)and serum creatinine(CR)were assessed to indicate glomerular filtration function,f MRI was performed to further confirm it.HE staining was used to evaluate the morphology and structure change of kidney.Oil red O staining was done to evaluate the distribution of lipid droplets in kidney.TEM to reflex the ultrastructural pathology change of glomerulus and renal tubule.3.Atgl-knockdown podocytes were established using sh RNA lentivirus transfection,F-actin staining,annexin V-Cy3 staining and TUNEL staining were down to observe the influence of down-regulation of Atgl on podocytes.4.Atgl-sh RNA podocytes were treated with NAC,an active oxygen scavenger.F-actin staining,annexin V-Cy3 staining and TUNEL staining were redone to determine whether intracellular ROS play a vital role in Atgl deletion-induced podocytes dysfunction.Results1.The body weights of Atgl-/-mice were significantly higher than those of Atgl+/+ mice at as early as8week-ageand progressively increased with age.Serum FFA,TG and cholesterol(TC)were significantly decreased in Atgl-/-mice.TG accumulation levels were dramatically increased in heart,liver and kidney of Atgl-/-mice compared with control group2.the urine ACR was significantly elevated in Atgl-/-mice;HE-staining showed a decrease in capsular space volume of kidney in Atgl-/-mouse;f MRI further showed a elevation of glomerular filtration rate in Atgl-/-mouse compare with Atgl+/+ mice.Oil red O staining hinted that lipid drops diffusedly distributed in glomerulus and renal tubules in Atgl-/-mice but not Atgl+/+ mice.TEM confirmed this phenomenon and further showed that in kidney of Atgl-/-mice,foot processes of podocytes were extensively effaced,microvilli also fell off and atrophied.The mitochondria in podocytes and renal tubule cells of Atgl-/-mice also showed collapse of internal cristae.3.Atgl knockdown promoted podocyte apoptosis accompanied by increased intracellular reactive oxygen species(ROS)and redistribution of F-actin fibre.These effects could be partially reversed by treatment with the antioxidant N-acetylcysteine(NAC).ConclusionTaken together,our findings indicate that Atgl plays important roles in maintaining normal lipid metabolism of the kidney and renal function.Atgl deletion in mice leads to ectopic deposition of TG in the kidney,and induces dysfunction of the glomerular filtration barrier and albuminuria.This may be achieved by F-actin rearrangement and mitochondria dysfunction,which induce footprocess fusion and podocytes apoptosis.ROS scavenger could effectively reduce the influence of ATGL down-regulated expression on F-actin rearrangement and apoptosis of podocytes. |
