The Mechanism Of LAMP1/2 Involved In Exosomes Mediated Renal Tubular Injury In Diabetic Kidney Disease And The Intervention Of Empagliflozin

Objective:Diabetic kidney disease is one of the main microvascular complications of diabetes,and it has become the main cause of end-stage renal disease.Therefore,it is very important to further explore the earlier biomarkers and effective therapeutic targets of diabetic kidney disease.Exosomes are microvesicles with a diameter of 30-100 nm containing a doublemembrane structure.At present,many studies have shown that urine exosomes are specific biomarkers with great potential for diagnosing diabetic kidney disease,and it is earlier than proteinuria.Furthermore,exosomes play important roles in the signal communication and interactive dialogue between cells.Therefore,exosome is largely helpful in diagnosing diabetic kidney disease and also involved in the pathogenesis of diabetic kidney disease.Lysosomes are responsible for processing proteins and lipids endocytosed in cells.Lysosomal membrane associated proteins 1 and 2(LAMP1/2)are abundant lysosomal membrane protein,which accounts for more than 50% of the total lysosomal membrane protein,and it is usually used as a lysosomal marker.Studies have shown that in disease states,lysosomal membrane associated proteins can be transferred to exosomes to reflect the early lysosome-related pathological changes.Furthermore,lysosomes play an important role in regulating the release of exosomes.Therefore,in this study,we detected the changes of urine exosomes and LAMP1/2levels in urine exosomes in patients with type 2 diabetes,subclinical diabetic kidney disease and early diabetic kidney disease to provide possible and effective biomarkers for the early diagnosis of diabetic kidney disease.And we further explored the mechanisms of LAMP1/2 in regulating exosomes release and participating in diabetic kidney disease.Methods:1.Patients with type 2 diabetes,subclinical diabetic kidney disease and early diabetic kidney disease were taken into the study.The basic information was recorded,blood and urine samples were collected and indicators were detected.Ultracentrifugation method was used to extract urine exosomes,and the levels of exosomal marker and LAMP1/2 were detected using Western blot method.2.Human renal tubular epithelial cells(HK-2)were used as the research object.It was treated with high level of glucose,free fatty acid and albumin,respectively,to explore the effects of the exosome release in cells.RT-q PCR method and Western blot was used to detect the m RNA expression and protein expression;Triglyceride detection reagent was used to detects triglyceride content in the exosomes;Lyso-Tracker-Red as the lysosomal probe was used to label lysosomes;exosomal inhibitor GW4869 was used to inhibit the pathway of exosomal release;lysosomal inhibitor Bafilomycin A1 was used to inhibit lysosome function.3.The fluorescent dye Di D was used to label exosomes.The laser confocal microscope was used to observe the uptake of exosomes by the HK-2 cells;the mitochondrial membrane potential detection kit was used to detect the damage of the mitochondrial membrane potential;the Annexin-FITC apoptosis kit was used to detect the number of cell apoptosis.4.We conducted loss-of-function or gain-of-function studies by transfecting small interfering RNA into cells to inhibit the expression of LAMP1/2 or transfecting plasmids to increase its expression to explore the mechanism of LAMP1/2 in regulating exosomes release and its own feedback damage.5.Empagliflozin was used to treat HK-2 cells to explore its function in regulating the release of exosomes and its self-feedback damage.Results:1.Compared with patients with type 2 diabetes,the number of urine exosomes and the content of LAMP1/2 in the exosomes in patients with subclinical diabetic nephropathy and diabetic nephropathy were increased(P <0.05),and they were positively correlated with UACR and renal tubular damage biomarkers(P <0.05).2.High level of glucose,free fatty acid and 1mg/ml albumin can also increase the degree of lysosomal dysfunction and the expression of LAMP1/2 in cells(P <0.05),thus increase the number of exosomes and the content of LAMP1/2 in exosome of HK-2 cells(P <0.05).3.High level of glucose can increase the content of cathepsin B in exosome of HK-2 cells(P <0.05);free fatty acids can increased triglyceride content in exosomes released by HK-2 cells(P <0.05);albumin(5mg/ml)can increased albumin content in exosomes released by cells(P <0.05).Exosomes carrying the contents can be further taken up by HK-2 cells and aggravate intracellular lysosomal dysfunction(P <0.05),and ultimately leads to damage of mitochondrial membrane potential and apoptosis(P<0.05).4.Overexpression of LAMP1/2 can improve the function of lysosomes under high glucose,free fatty acid and albumin,thereby reducing the number of exosomes released by cells(P <0.05)and the cathepsin B,triglycerides and albumin in the exosomes(P<0.05),thereby improving the self-feedback damage of exosomes.Inhibition of LAMP1/2 has the opposite effect on the regulation of exosomes.5.Empagliflozin can balance the number and function of lysosomes by regulating LAMP1/2(P <0.05),thereby reducing the number of exosomes and cathepsin B,triglycerides and albumin in exosomes(P <0.05)under high glucose,free fatty acid and albumin(P <0.05),ultimately improve the self-feedback damage of exosomes.Conclusion:1.The number of urine exosomes and the level of LAMP1/2 in the exosomes may be effective biomarkers for the early diagnosis of diabetic kidney disease.Furthermore,they may be specific markers to reflect the lysosomal dysfunction in renal tubular epithelial cells.2.High glucose can increase the content of cathepsin B in exosomes;free fatty acid can increase the content of triglycerides in exosomes;albumin can increase the content of albumin in exosomes.Exosomes and their contents can be further taken up by HK-2 cells,and ultimately leads to cell mitochondrial membrane potential damage and cell apoptosis.LAMP1/2 may be common target.3.Empagliflozin can regulate the expression of LAMP1/2 in HK-2 cells treated with high glucose,high fat and high protein,thereby restoring the balance of lysosome function,reducing the number of exosomes released and their contents,and finally improve the self-feedback damage of exosomes.