| Objective: Acute hyperglycemia can induce a series of pathophysiological changes caused by acute metabolic disorders like diabetic ketosis and hyperosmolar hyperglycemia;in addition,acute hyperglycemia can also suppress insulin secretion function of islet β-cells transiently which is called “acute hyperglycemic toxicity”.Recent studies have suggested that acute hyperglycemia can also lead to “acute renal toxicity”.We had observed the “toxic effects” of acute hyperglycemia on the kidney of healthy rats using hyperglycemic clamp technique in our previous work.Our results showed that hyperglycemia keeping around 16.7mmol/L for 6 hours can damage renal tubular epithelial cell morphology and reabsorption function,with mitochondria injured and oxidative stress activated,but the mechanism is still not clear.Cells clear away damaged organelles and other components through autophagy which plays a very important role in acute organ injury.The insufficiency of mitophagy can lead to ROS accumulation in cells which will aggravate cell injury.On the basis of our previous research,we will further explore:1.injury-concentration relationship of acute hyperglycemia induced renal tubular injury in healthy rats.2.Reversibility of renal tubular injury induced by acute hyperglycemia in healthy rats.3.The role of mitophagy and AMPK-m TOR regulation pathway in acute hyperglycemia induced renal tubular injuryMethods:1.Healthy SD rats were selected as the research objects:(1)Study on the injury-concentration relationship of acute hyperglycemia induced renal tubular injury: rats were divided into four groups randomly,control group,hyperglycemia group A(11.1mmol/L),hyperglycemia group B(16.7mmol/L)and hyperglycemia group C(25.0mmol/L),10 in each group.(2)Study on the reversibility of acute hyperglycemia induced renal tubular injury: rats were divided into four groups randomly,control group(normal saline),hyperglycemia group B(16.7mmol/L 6h,24 h death),hyperglycemia group B1(16.7mmol/L 6h,48 h death)and hyperglycemia group B2(16.7mmol/L 6h,72 h death),10 in each group.2.Rats were undertaken internal jugular venipuncture 5 days before hyperglycemic clamp.During the hyperglycemic clamp,we kept blood glucose of rats in hyperglycemia group by continuous pumping of 50% glucose solution for 6 hours.And we infused saline at the same speed in rats in control group.3.Serum and kidneys were collected after the rats were killed.We observed the glomerular and tubular morphology changes of healthy rats using both optical microscope and transmission electron microscopy.We observed the autophagosome in the renal tubular epithelial cells using transmission electron microscopy.Biochemistry method was used to test the changes of serum creatinine,urea nitrogen,creatinine clearance rate,urine albumin(UMA)collected within 24 hours,urine β2-MG,NAG and GAL after the hyperglycemic clamp.ELISA was used to test urine NGAL and Cys C changes which represent renal tubular damage.Western blot and RT-PCR was used to test the level of P62,the ratio of LC3-II/LC3-I,the level of BNIP3L/Nix which represent activity of autophagy or mitophagy.Western blot and RT-PCR was used to test level of sodium glucose transporter SGLT-2.Western blot was used to test the phosphorylation levels of AMPK and m TOR which were the regulatory pathway of autophagy or mitophagy.4.Rat renal tubular epithelial cells(NRK-52 E cells)were incubated using different concentrations of glucose(5.5mmol/L,11.1mmol/L,16.7mmol/L,25.0mmol/L)for 6h.MTT method was used to test the cell viability.Western blot and RT-PCR were used to test the level of P62,the ratio of LC3-II/LC3-I and BNIP3L/Nix which represent activity of autophagy or mitophagy.Western blot and RT-PCR was used to test level of sodium glucose transporter SGLT-2.Western blot was used to test the phosphorylation levels of AMPK and m TOR which were the key molecules of regulatory pathway of autophagy or mitophagy.5.Further investigation of regulatory pathway of mitophagy: NRK-52 E cells were divided into four groups: control group(5.5mmol/L),hyperglycemia group B(16.7mmol/L),AMPK activator group(16.7mmol/L glucose + AICAR)and m TOR inhibitor group(16.7mmol/L + rapamycin).Cells were incubated for 6h,and total RNA and protein were extracted.Western blot and RT-PCR was used to test the level of P62,the ratio of LC3-II/LC3-I and BNIP3L/Nix which represent activity of autophagy or mitophagy.Western blot and RT-PCR was used to test level of sodium glucose transporter SGLT-2.Western blot was used to test the phosphorylation levels of AMPK and m TOR which were the regulatory pathway of autophagy or mitophagy.Results:1.We set up the acute hyperglycemia model in healthy rats successfully,the blood glucose was maintained around 11.1mmol/L,16.7mmol/L and 25.0mmol/L respectively in each hyperglycemic group,and the blood glucose of rats in control group was maintained between 4-6 mmol/L.2.We saw acute hyperglycemia with the level of 11.1mmol/L could lead to swelling of tubular epithelial cells under optical microscope,and local foot process merging together,windows of endothelial cells enlargement,some nucleus apoptotic characteristics accompanied by swelling of endothelial cells,unclear internal composition of epithelial cells and mitochondria accompanied by autophagosome and autolysosome increasing in three different concentration groups of acute hyperglycemia under transmission electron microscope,and the injury severity aggravated gradually with the increase of hyperglycemia concentration.3.After withdraw the intervention of hyperglycemia for 24 hours,we saw the swelling of tubular epithelial cell,the lumen stenosis of tubular were more obvious under optical microscope,which relieved gradually after withdraw the intervention for 48 h and 72 h,but not fully restored to normal 72 h later(still slight swelling in the renal tubules).4.Acute hyperglycemia with all of the three concentrations(11.1mmol/L,16.7mmol/L and 25.0mmol/L)could increase the level of UMA and the level of urine NAG,NGAL,GAL,Cys C and β2-MG which represent tubular epithelial cell injury in 24 hours of healthy rats(P < 0.05),but no obvious changes of serum creatinine,urea nitrogen,creatinine clearance rate were observed(P > 0.05).5.The reversibility of renal injury was further observed through withdraw the intervention of hyperglycemia(16.7mmol/L)for 24 h,48h and 72 h respectively.We saw the level of UMA was increased in 24 h later of healthy rats compared with the control group(32.37±3.65 vs 10.25±0.84 μg/24 h,P < 0.05),but decreased in 72 h later(12.15±1.34 vs 32.37±3.65 μg/24 h,P < 0.05).There was no any change of serum creatinine,urea nitrogen and creatinine clearance rate with the time going on(P > 0.05).The level of urine NAG,NGAL,GAL,Cys C and β2-MG which represent tubular epithelial cell injury were also increased in 24 h later and decreased in 72 h later,but not fully restored in 72 h later(NAG 10.58±2.38 vs 4.85±3.75 U/L,P > 0.05,NGAL 1.63±0.56 vs 0.72±0.11 ng/m L,P > 0.05,GAL 22.49±5.98 vs 16.50±0.71 U/L,P > 0.05,Cys C 123.01±17.24 vs 24.93±12.69 ng/m L,P < 0.05,β2-MG 0.043±0.010 vs 0.026±0.011 mg/L,P > 0.05).6.Compared with that in control group,increased the expression of P62 and BNIP3L/Nix,decreased the ratio of LC3-II/LC3-I which represent the degree of autophagy,increased expression of SGLT-2 and the phosphorylation level of m TOR,but decrease phosphorylation level of AMPK in healthy rats kidney were observed in the three hyperglycemic groups.7.Acute hyperglycemia can upregulate P62,BNIP3L/Nix expression and down-regulation LC3-II/LC3-I ratio which represent the degree of autophagy,increase phosphorylation level of m TOR but decrease phosphorylation level of AMPK in NRK-52 E cell.8.Compared with that in the acute hyperglycemia group(16.7mmol/L),AMPK activator and m TOR inhibitors groups could increase the ratio of LC3-II/LC3-I,decrease the expression of P62 and BNIP3L/Nix which represent the degree of autophagy in NRK-52 E cell(P < 0.05).Conclusion:1.Acute hyperglycemia can lead to renal morphology and functional injuries,and began at least at the level of 11.1mmol/L,and behaves in a dose-dependent manner.Tubular injuries turned out to be more sensitive.2.The renal tubular injury induced by acute hyperglycemia was reversible.The renal tubular injury was gradually restored with prolongation of time after withdraw the intervention of acute hyperglycemia.3.Mitophagy were inhibited in epithelial cells of damaged renal tubules,which was related to hyperglycemia concentration.4.AMPK/m TOR pathway was involved in the regulation of mitophagy inhibited by acute hyperglycemia induced renal tubular epithelial cell injuries. |
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