Molecular Markers Diagnosis And MicroRNA-7977 /SIRT3/SOD2/ROS Signal Pathway In "IGT Nephropathy"

Objective:The incidence of diabetes has increased rapidly in recent years.Diabetic nephropathy is a microvascular complication of diabetes and it is also primary basic cause of end-stage renal disease.At present,microalbuminuria(MAU)is the main basis for diagnosing early diabetic nephropathy.However,many new studies have shown that its sensitivity and specificity are not ideal.Therefore,it is of great significance to find new biomarkers to diagnose earlier kidney damage.Prof.Pan Changyu et al.found that 13.1% of patients with pre-diabetes have had MAU.Our group has previously observed significant structural and functional impairment of the kidneys in rats with impaired glucose tolerance.In contrast to "diabetic nephropathy",we named the kidney disease at this stage as "IGT nephropathy".We further studied that insulin resistance and hyperinsulinemia are important causes of "IGT nephropathy".The concept of "IGT nephropathy" has provided a new perspective for the early diagnosis of diabetic nephropathy.Although the renal damage in IGT was observed in animal experiments,it needs to be further verified in clinical patients.Besides,the diagnosis basis and exact pathogenesis need to be further explored.A variety of biomarkers are valuable for the early diagnosis of diabetic nephropathy in current studies.The inflammatory factors such as TNF?,TNFR1,TNFR2,MCP-1,C-reactive protein and ceruloplasmin can be used as molecular biomarkers for the early diagnosis of diabetic nephropathy.At the same time,cytokines such as FGF21 and adiponectin which can regulate the metabolism of glucose and lipid have already changed during the onset of diabetic nephropathy.In recent years,a type of short-chain and non-coding RNA named micro RNA has become an important factor to regulate gene expression.It can participate in the regulation of numerous gene expressions by binding to specific sites of m RNA.A large number of studies have found that many kinds of micro RNAs are involved in the development of insulin resistance,diabetes and diabetic nephropathy.Its value in the early diagnosis of diabetic nephropathy is of increasing concern.Have these molecular markers changed in the stage of "IGT nephropathy" ? Furthermore,What is the role in the pathogenesis of "IGT nephropathy" ?The aim of this study was to observe the change of general data,biochemical and renal impairment indicators in normal control(NC),impaired glucose tolerance(IGT),IGT nephropathy(IGTN),type 2 diabetes(T2DM)and type 2 diabetic nephropathy(T2DN)in the population based on previous animal and cell experiments and further validate the occurrence of renal injury in a clinical perspective.At the same time,we further studied the changes in serum molecular markers and urinary micro RNAs aimed at finding more sensitive and specific markers for diagnosis of "IGT nephropathy" and exploring the possible pathogenesis of "IGT nephropathy".Method:1.We collected the basic information and samples of serum and urine among the population.Liver and kidney function,blood lipids and blood glucose were tested by biochemical methods;Insulin was tested by radioimmunoassay;B-ultrasound scanner was used for measuring kidney size;microalbumin(UMA),N-acetyl-?-glucosaminidase(NAG),retinol-binding protein(RBP),?2-microglobulin(?2-MG),transferrin(TF)and immunoglobulin G(Ig G)were tested by biochemical methods;Enzyme-linked immunosorbent assay(ELISA)was used to detect urinary cystatin C(Cys C)and neutrophil gelatinase-associated apolipoprotein(NGAL);According to these works above,we initially identify petients with "IGT nephropathy".2.Tumor necrosis factor-?(TNF?),tumor necrosis factor receptor 1(TNFR1),tumor necrosis factor receptor 2(TNFR2),adiponectin,monocyte chemotactic protein 1(MCP1),ceruloplasmin,fibroblast growth factor 21(FGF21)and highly sensitive C-reactive protein were tested by ELISA.The ROC curves were generated to evalute the sensitivity and specificity of the index in the diagnosis of diabetic nephropathy.We also observed changes of these biomarkers in patients with "IGT nephropathy".3.Urine microRNA profile of patients with "IGT nephropathy" was established by gene sequence technology.Signaling pathway enrichment analysis was used to integrate the signal pathways that differential micro RNA might participate in.We expand the sample size in the population to verify differential micro RNA in the microarray.4.Renal tubular epithelial cells(HK-2)were used as research objects.It was divided into blank control group,low-intensity insulin intervention group(5ng/ml)and high-dose insulin intervention group(50ng/ml).mir-7797,SIRT3,Ac-SOD2 and SOD2 were detected by RT-q PCR,western blot and immunofluorescence.The fluorescence probe method was used to determine the level of reactive oxygen species(ROS)in the cells.We further overexpressed or inhibited mir-7977 and detected its downstream signaling pathway.The dual luciferase reporter assay was used to verify the direct regulation of mir-7977 on SIRT3.5.The cells were transfected with small interfering RNA(si RNA)in order to reduce the expression of SIRT3.RT-q PCR,western blot and immunofluorescence were used to detect the expression of Cubilin and albumin endocytosis.Laser confocal microscopy was used to examine endocytosis of fluorescently labeled albumin on cells.Result:1.Comparison of glucose and Insulin: Compared with NC group,the 2 hours postprandial blood glucose was increased in IGT and "IGT nephropathy";compared with groups of NC and IGT,the level of 2-hour postprandial insulin was increased in groups with "IGT nephropathy" and T2DM(P<0.05);the HOMO-IR was increased gradually among these five groups.Compared with the first three groups,the HOMO-IR in the T2 DM and DN groups were increased(P<0.05).2.Compared with NC group,the biomarkers of glomerular damage including albumin,transferrin,immunoglobulin G and tubular damage including NAG,RBP,GAL,NGAL and cystatin C in "IGT nephropathy" were all increased(P<0.05).In patients with "IGT nephropathy",the long diameter of kidney and glomerular filtration rate were increased.3.Comparison of other serum molecular markers that are biomarkers of early diabetic nephropathy: TNFR1,TNFR2,TNF?,MCP-1,ceruloplasmin,adiponectin,highly sensitive C-reactive protein,and FGF21 were gradually increased in the groups of NC,IGT nephropathy and DN,but there was no significant change in the groups of IGT and T2 DM.Compared with IGT,serum TNFR2,MCP-1 and ceruloplasmin were increased in "IGT nephropathy"(P<0.05),and were further increased in DN(P<0.05).Compared with the first four groups,FGF21,TNF?,TNFR1,adiponectin,and high-sensitivity C-reactive protein were elevated in patients with DN(P<0.05).4.There were many changes in the microRNA content in urine of petients with "IGT nephropathy".It revealed that differential micro RNAs may participate in the pathogenesis of "IGT nephropathy" through signaling pathways such as m TOR,phosphatidylinositol,vascular smooth muscle contraction,Notch signaling pathway and NOD-like receptors by analysising of KEGG signaling pathway enrichment.Verification of differential micro RNAs in microarray results: mir-7977: Compared with the NC group,"IGT nephropathy" and DN groups were both elevated(P <0.05);mir-5100: IGT and T2 DM groups were higher than NC group(P<0.05),but diabetic nephropathy group was lower than IGT and T2 DM group(P<0.05).5.Effect of high concentration of insulin on mir-7977/SIRT3/SOD2/ROS signaling pathway: In the group of high concentration of insulin,the expression of mir-7977 was significantly increased(P<0.05),SIRT3 were decreased(P<0.05),and Ac-SOD2/SOD2 and ROS were increased(P<0.05).Compared with the control group,SIRT3 in mir-7977 mimics group was further decreased,Ac-SOD2/SOD2 and ROS were increased(P<0.05);SIRT3 in mir-7977 inhibitor group was increased,and Ac-SOD2/SOD2 and ROS were both decreased(all P<0.05).6.Effect of SIRT3 on Cubilin and endocytosis of albumin: Compared with the control group,the m RNA and protein levels of Cubilin in the low-dose insulin group were both increased(P<0.05).Compared with the normal insulin group,the m RNA and protein levels of Cubilin in the groups of high concentration insulin were decreased(P<0.05).SIRT3 si RNA can decreased the m RNA and protein expression of Cubilin and endocytosis of albumin in both normal and high concentrations of insulin(P<0.05).Conclusion:1."IGT nephropathy" was further confirmed in clinical patients.Patients with "IGT nephropathy" have different degrees of glomerular and tubular damage.Glomerular injury is mainly manifested by elevated urinary microalbumin,transferrin,and immunoglobulin G,and renal tubular injury is mainly manifested by elevated urinary NAG,RBP,GAL,NGAL and cystatin C.In patients with "IGT nephropathy",the long diameter of kidney and glomerular filtration rate were increased.2.Serum TNFR2,MCP1,ceruloplasmin were elevated in patients with "IGT nephropathy",and the concentration of TNFR2 and MCP1 was positively correlated with "IGT nephropathy".These biomarkers may be helpful to identify and diagnose"IGT nephropathy".3.Hyperinsulinemia after glucose overload is closely related to the pathogenesis of"IGT nephropathy".4.Compared with the NC group,there were many up-regulated and down-regulated micro RNA in the urine of the "IGT nephropathy" group.Different micro RNA may participate in the m TOR signaling pathway,phosphatidylinositol signaling system,vascular smooth muscle contraction,Notch signaling pathway,NOD-like receptor signaling pathway,glycosaminoglycan biosynthesi and aldosterone-regulated sodium reabsorption by regulating the expression of its target genes thus participating in the occurrence and development of "IGT nephropathy".5.mir-7977 were elevated in the "IGT nephropathy" group and may be molecular markers for early diagnosis of "IGT nephropathy".6.High concentration of insulin can active the mir-7977/SIRT3/SOD2/ROS signaling pathway,causing oxidative stress damage and decreased expression of Cubilin which mediates albumin endocytosis in renal tubular epithelial cells,resulting in the reduced reabsorption of albumin.