The Role And Mechanism Of NOD2 In Diabetic Kidney Disease Glomerular Injury

BackgroundDiabetic kidney disease(DKD)has become one of the most important causes of end-stage renal disease worldwide.Currently,there is a lack of effective therapeutic interventions for DKD.Exploring the molecular mechanism of the occurrence and development of DKD is expected to provide more effective targets for treatment.Studies have shown that DKD is also a "podocyte disease",which is clinically characterized by proteinuria.In pathology,there are fusion and detachment of foot processes,and even podocyte apoptosis,and the filtration barrier structure is abnormal.The hyperglycemic environment affects the protein expression of podocytes.It also destroys the skeletal structure of the podocytes and the shape of the foot processes,leading to filtration dysfunction and even podocyte apoptosis.Restoring the physiological structure and function of podocytes is of great significance to the treatment of DKD.But so far,the molecular mechanism underlying the morphological disorder of podocytes in DKD is still unclear.DKD is a chronic inflammatory disease caused by metabolic disorders,indicating that microinflammation is a common mechanism of diabetic vascular complications.Recent studies have shown that immune cells activating innate immune responses and intrinsic kidney cells play a key role in triggering and maintaining inflammation.Zinc finger transcription factor(Snail)is one of the important upstream molecules involved in this process,alleviating inflammation and fibrosis.Pattern recognition receptors(PRR)are a family of innate immune system receptors,which have been extensively studied in recent years and play an important role in renal immunity and inflammation.TLR(Toll-like receptor)and NLR(nucleotide-binding oligomerization domain-like receptor)are the two most important subfamilies of PRR.TLR is a membrane-bound receptor,and NLR is distributed in the cytoplasm.NOD is a type of NLR.Studies have found that NOD2,a subtype of NOD,is not only expressed in inflammatory cells,but can also be detected in the intrinsic kidney cells.However,research on the role of NOD2 in DKD is still insufficient.The way that NOD2 affects the progression of DKD is unclear.Therefore,our research aims to explore the molecular mechanism of NOD2 causing DKD kidney damage and provide a more effective target for the treatment of DKD.Objective1.Observe the expression level of NOD2 in kidney tissue and serum of patients with Diabetic kidney disease.2.Construct DKD model in NOD2-knockout mice to further verify the changes in NOD2 expression and clarify its effects on Diabetic kidney disease.Methods1.1 The human serum samples and tissue samples used in the experiment are from the biological sample bank of the First Affiliated Hospital of Zhengzhou University,which met the sampling requirements of the biological sample center and are used for immunohistochemistry and ELISA experiments.1.2 Eight-week-old mice with C57BL/6N genetic background were purchased from Beijing Bioset Corporation.The NOD2 gene knockout mice were bred and provided by Biocytogen.All mice are kept in the Animal Experiment Center of Zhengzhou University.1.3 Divide mice into 4 groups:Con group,DKD group,NOD2-/-Con group and NOD2-/-DKD group.Mice in the DKD group and NOD2-/-DKD group were modeled by streptozotocin(STZ)and high-fat diet,while the Con group and NOD2-/-Con group were fed a normal diet.At 4 weeks,mice in the DKD group and NOD2-/-DKD group were induced by intraperitoneal injection of 50 mg/kg body weight of STZ.For 5 consecutive days.The Con group and NOD2-/-Con group were injected with the same amount of normal saline.One week after the last injection of STZ,a blood sample from the tail vein was collected and the fasting blood glucose level was measured using a glucose analyzer.Mice with blood glucose level≥16.7mmol/L are considered to be successful.1.4 After the model was established,feeding continued for another 12 weeks,and blood glucose was measured every 4 weeks.One day before the end of the experiment,urine samples were collected.When the mice were sacrificed,the kidney tissues of the mice were stained with HE staining,Masson staining,PAS staining,and immunofluorescence double-labeling staining.Part of the tissue is frozen at-80 degrees Celsius and used for subsequent Western blot experiments for tissue protein determination.Another part was sent to the Electron Microscopy Pathology Room of the First Affiliated Hospital of Zhengzhou University,where the nephropathologists made the electron microscope section specimens,and under the guidance of the nephropathologists,the electron microscope photos of the mouse kidney were taken.Results1.NOD2 is highly expressed in the tissues of patients with Diabetic kidney disease.2.The expression of NOD2 in the serum of patients with Diabetic kidney disease was significantly higher than that of the control group.3.NOD2 is highly expressed in Diabetic kidney disease mice,and the knock-out mice are successfully modeled.4.Knockout of NOD2 gene can reduce cortical and medullary fibrosis in Diabetic kidney disease5.Reducing the expression of NOD2 can improve the damage of Diabetic kidney disease to the intrinsic cells of the kidney.6.Diabetic kidney disease reduces the expression of intrinsic cell-related proteins in the kidney and increases the fibrotic phenotype protein.Knockout of the NOD2 gene improves this result.7.The results of immunofluorescence suggest that Snail1 is involved in glomerular damage and fibrosis.NOD2 regulates Snail through the IKK/NF-κB pathwayConclusionNOD2 regulates Snail-mediated fibrosis of Diabetic kidney disease through the IKK/NF-κB pathway,which can aggravate the kidney damage of Diabetic kidney disease.Therefore,it is expected that blocking NOD2 expression will become a therapeutic target for Diabetic kidney disease.