The Damage Of Proximal Tubule Caused By Beta Hydroxybutyric Acid And Its Mechanism

Objective:Diabetic ketosis and diabetic ketoacidosis are severe acute complications of diabetes,which not only cause metabolic disorders in diabetic patients but may also cause acute kidney injury^[1].Such patients are often encountered in clinical practice:patients with newly diagnosed diabetes mellitus（DM）with positive urine protein will be diagnosed as"diabetic nephropathy",especially those with ketosis.In our previous research,our group found that acute hyperglycemia has"nephrotoxic damage",which mainly shows different degrees of damage to the structure and function of the glomerulus and tubules,especially in renal tubules^[2].So,whether excessive ketone bodies will damage the kidneys when diabetic ketosis?If so,what’s the mechanism of it?These issues deserve attention.The purpose of this study was to investigate whether excessive ketone bodies in diabetic ketosis can cause kidney damage,and to preliminary explore its possible mechanism of action.Method:1、Taking the non-diabetic ketoacidosis and diabetic ketoacidosis patients as the research object,collect general information,detect fasting blood glucose,glycated hemoglobin,liver function,renal function,electrolytes,blood lipids and other basic biochemical information,detect urinary ketone bodies,and divide the population into2 groups by ketone bodies,the groups are diabetic non-ketosis group（NDK）,diabetic ketone body group（DK）,and normal control group（NC）are set up simultaneously.Then detecting urine kidney injury biomarkers respectively,such as urine microalbumin（UMA）,ACR（UMA/u Cr）,transferrin（TF）,immunoglobulin G（Ig G）,tubular injury biomarker:β2 microglobulin（β2-MG）,retinol binding protein（RBP）,N-acetyl-β-D-glucosidase（NAG）,galactosidase（GAL）,all Corrected with urinary creatinine（u Cr）and analyzed differences among the groups.2、Taking the diabetic ketosis group as the research object,collecting patient data before and after diabetic ketosis correction,and detecting renal biomarkers in urine:microalbumin（UMA）,ACR（UMA/u Cr）,transferrin（TF）,immunoglobulin G（Ig G）,β2 microglobulin（β2-MG）,retinol binding protein（RBP）,N-acetyl-β-D-glucosidase（NAG）,galactosidase（GAL）,analysis of ketosis correction Differences in urinary and renal injury markers before and after.3、Taking human proximal renal tubular cells（HK-2）as research objects,using different concentrations ofβ-hydroxybutyric acid（0～4mmol/L）and acetoacetic acid（0～16mmol/L）to interfere with HK-2 cells.Then observing the expression of Megalin,and fluorescein isothiocyanate-labeled albumin（FITC-BSA）endocytosis.4、Human renal tubular epithelial cells（HK-2）were used as the research object,and different concentrations ofβ-hydroxybutyric acid（0～4mmol/L）were used to interfere with HK-2 cells.Observing the changes of Megalin and its upstream AKT and DAB2.5、Overexpression or inhibition of AKT phosphorylation level,detecting the changes in downstream signal pathways DAB2 and Megalin,and detecting the fluorescence intensity of HK-2 cells endocytosis with fluorescein isothiocyanate-labeled albumin.6、Taking human renal tubular epithelial cells（HK-2）as research objects,to overexpress DAB2 and observe the expression of Megalin and its effect on the endocytosis of albumin in HK-2 cells.Result:1、Compared with the NC and NDK groups,the glomerular filtration rate（e GFR）in the DK group increased significantly（112.49±13.61 VS 117.31±25.21 VS164.72±36.25 ml/min?1.73m²,P value<0.05）;Among on NC group,NDK group and DK group,ACR was significantly increased（0 VS 0 VS 39.5%,P value<0.05）,and urine ACR,TF,Ig G,β2-MG,RBP,NAG,GAL levels gradually increased（P value<0.05）,and the abnormal rise rate of urine TF,Ig G,β2-MG,RBP,NAG,GAL increased gradually（P value<0.05）.2、Compared with diabetic ketosis,the group of diabetic ketosis correction is no significant difference in blood glucose（16.68±4.88 VS 11.12±3.67mmol/L,P value>0.05）,the kidney injury index UMA was significantly reduced[3.2（1.40,15.85）VS 1.80（1.10,3.25）mg/L,P value<0.05],and urine TF,Ig G,β2-MG,RBP Significantly reduced（P value<0.05）,and it has no significant diffrence in urine NAG,GAL（P value>0.05）.3、As the concentration ofβ-hydroxybutyric（BOHB）increased,the amount of albumin untaken by HK-2 cells decreased.The amount of endocytosed albumin gradually increased in the PH control group corresponding to the p H value of the culture medium intervened withβ-hydroxybutyric acid.At a constant p H of 7.4,as the concentration ofβ-hydroxybutyric increased,the amount of albumin in endocytosis decreased,the expression of Megalin decreased in HK-2 cells,and the concentration of 4mmol/L was statistically significant（P value<0.05）.4、Compared with the BOHB intervention group,the expression level of DAB2increased significantly（P value<0.05）,and The amount of albumin was also significantly increased in HK-2 cells in the BOHB+AKT activator group;the expression level of DAB2 was significantly reduced（P value<0.05）,and the amount of albumin was also significantly decreased in HK-2 cells in the BOHB+AKT inhibitor group.5、Compared with the BOHB group,the expression level of Megalin increased significantly,and the amount of albumin in HK-2 cells also increased significantly in the BOHB+DAB2 overexpression group.6、With Acetic acid（Ac Ac）intervention concentration increased,the expression of Megalin in proximal renal tubular epithelial cells decreased,the amount of albumin in HK-2 cells decreased accordingly.When the concentration reached 16 mmol/L,they were statistically difference（Considering that 16mmol/L is too high relative to the physiological concentration,the experiments related to the AKT/DAB2 signaling pathway of acetoacetate were not continued）.Conclusion:1、It has been clinically verified that patients with diabetic ketosis can have different degrees of renal damage,in addition to microalbuminuria,there can also be some renal tubular damage.After the ketosis correction patients’s microalbuminuria and renal tubular injury-related markers recover to some extent.2、As the main component of the ketone body,high concentration ofβ-hydroxybutyric acid down-regulates the expression of the endocytic receptor protein Megalin throμgh the AKT/DAB2 pathway,and inhibits proximal tubμlar endocytosis.