The Expression Of HMGN1/TLR4 And Association With Diabetic Kidney Disease Explored In Mouse Models

Objective:To determine the renal expression of high-mobility group nucleosome binding protein 1(HMGN1)and toll-like receptor(TLR4)in diabetic kidney disease(DKD)mouse models,and explore the association between HMGN1/TLR4 and renal function as well as pathology.The study provides a theoretical basis for the application of HMGN1/TLR4 pathway in DKD immunotherapy.Methods:In vivo:1.Animal models and groups:(1)Type 1 diabetic nephropathy(T1DN):15 C57BL/6 mice were randomly divided into 3 groups:NC group;T1DN group[a single 150 mg/kg dose of streptozocin(STZ)intraperitoneally injection];T1DN+INS group[STZ induced diabetic model followed by intraperitoneally injected with 1U/d insulin for 8 weeks].All group mice were sacrificed for blood,urine and kidney tissue collection after drug treatment.(2)Type 2diabetic nephropathy(T2DN):20 C57BLKS/J mice were selected as WT group;40C57BLKS/J-db/db mice were randomly divided into 2 groups:T2DN group and T2DN+INS group(1U/d insulin).The blood,urine and kidney tissue samples were collected from the mice at 6,8,12,16 weeks of age respectively.2.Routine biochemistry index:fasting blood glucose,kidney index and urea albumin creatinine ratio(UACR)were measured.3.Renal pathological changes:hematoxylin-eosin(HE)and Periodic acid-Schiff(PAS)staining were used to show the changes of renal tissue structure and determine the mesangial index.4.The expression of HMGN1 and TLR4 mRNA and protein in kidney tissues:detected by in situ hybridization(ISH)and immunohistochemistry(IHC)and the correlation with other parameters were further analyzed.In vitro:1.Groups:NRK-52E(ATCC~?CRL-1571?)cells were grouped as follows:Control group(5mmol/L glucose);High glucose group(25mmol/L glucose).2.The expression of HMGN1,TLR4 mRNA and protein in cells:detected by ISH and western blot after 72 hours of treatment.Results:In vivo:1.Compared with control groups,the blood glucose,kidney index,UACR and mesangial index were significantly increased[(8.57±0.70)vs(24.28±1.944 mmol/L,(1.44±0.02)vs(1.70±0.09)mg/g,(97.69±10.68)vs(697.67±168.78)?g/mg,P<0.05];characteristic lesions are shown with mesangial expansion,renal tubules with different degrees of injury and tubulointerstitial infiltration with inflammatory cells;the expression of HMGN1 and TLR4 in kidney tissue was up-regulated in T1DN groups(P<0.05);a significant positive correlation was showed among the expression of HMGN1,TLR4 and mesangial index(r=0.337,0532,0.369,0.366,0.574,0.826,0.359,0.3175,P<0.05);no significant changes were observed in the insulin treatment group(P>0.05).2.In T2DN group,both biochemical and pathological indicators suggested the features of diabetic nephropathy in T2DN group,HMGN1 expression in renal tissue was increased at 6 weeks of age and reduced at 8 weeks of age in the kidney tissue,TLR4 expression maintained high level along with aging,when compared with WT group(P<0.05).Moverover,the depressed level of HMGN1 was negatively correlated with TLR4 expression,UACR,mesangial index,respectively(r=-0.450,-0.515,-0.622,-0.295,P<0.05).In vitro:The decreased expression of HMGN1 and increased expression of TLR4 were detected in high glucose group when compared with control group(P<0.05).Conclusion:1.Increased expression of HMGN1/TLR4 was detected to be involved in T1DN progression;reduced expression of HMGN1,persistent high expression of TLR4 was observed in early T2DN as well as in high glucose induced NRK cells instead.2.Different role of HMGN1 could be implied in the pathological mechanisms between T1DN and T2DN.3.Hyperglycemia could be implicated in the reduced level of HMGN1 in T2DN;potential mechanisms,but not hyperglycemia,primarily contributing to promote the increased level of HMGN1 in T1DN was existed.