The Role Of Osteoprotegerin In Type 2 Diabetic Kidney Disease And The Clinical Application Of Fracture Risk Assessment Tool(FRAX)

Background:Osteoporosis is mainly characterized by fragility fracture.Osteoprotegerin(OPG),as a marker of bone formation,mainly inhibits osteoclast differentiation and activity through OPG/RANK/RANKL system.OPG is also considered to have some extraosseous effects,such as participation in cardiovascular and immune system diseases,and antitumor effects,but it remains to be determined.In addition,the role of OPG in metabolic diseases is not clear.Some studies believed that OPG could protect β cell,however,the role of OPG in the pathogenesis of diabetes and diabetic complications needs further exploration,furthermore,OPG may even become new biomarkers or therapeutic targets.Osteoporotic fracture is one of the complications which affects patients’ quality and safety of life seriously.It has multiple risk factors.Among them,thyroid dysfunction and type 2 diabetes kidney disease may aggravate the risk of fracture due to their comorbidity.Consequently,how to prevent the comorbidity of these two diseases and postpone their progression is an urgent problem to be solved.In addition,the fracture risk assessment tool(FRAX)recommended by WHO,can predict the probability of major osteoporotic fracture and hip fracture in the next 10 years.However,the clinical factors involved are limited,and the fracture risk of Chinese population may be underestimated,therefore,it is of certain significance to explore the application characteristics,applicable objects of FRAX and the influence by other factors(such as biochemical indicators of bone metabolism)in a large sample population.In this paper,we explore the role of OPG in type 2 diabetes kidney disease by clinical research and animal experiment.The interaction between thyroid dysfunction and type 2 diabetes kidney disease is discussed.The application characteristics of FRAX in general population and the correlation with biochemical indicators of bone metabolism are analyzed.The aims of this study are to provide a new evidence for the endocrine role of bone in metabolic diseases,and to provide a new idea for guiding the early intervention of osteoporotic fracture.Part one:OPG level is associated with risks of DKD and DKD progression in patients with type 2 diabetesObjective:(1)To analyze the correlation between serum OPG level and DKD in patients with type 2 diabetes.(2)To analyze the correlation between serum OPG level and risk of DKD progression type 2 diabetes.Methods:(1)All subjects were patients with type 2 diabetes.They were routinely hospitalized in the Department of Endocrinology and Metabolism of the Third Hospital of Nanchang between February 2018 and November 2020.All subjects participated in the study voluntarily and signed written the informed consent form at admission.(2)Medical history collection,physical examination,and blood and urine samples testing were performed.(3)Staging of CKD(G1-G5)were defined by eGFR,albuminuria grouping(normal,microalbuminuria,massive albuminuria)were divided by UACR,and risk of CKD progression(low,moderate,high or very high risks)were recommended by the Kidney Disease:Improving Global Outcomes(KDIGO).Results:(1)452 patients were included in the study.The average level of OPG was 976.49± 589.10 pg/ml.(2)The level of OPG decreased gradually with the increase of the aggravation of albuminuria and CKD progression.No differences were found between OPG levels and stages of CKD.(3)After the adjustment,each SD increase in OPG level could reduce the risks of massive albuminuria and the high risk of DKD progression by 36%and 29%,respectively.No correlations were found between OPG and stages of CKD.Conclusions:In patients with type 2 diabetes,elevated serum osteoprotegerin(OPG)is an independent protective factor for albuminuria and risk of CKD progression.Part Two:The role of osteoprotegerin in diabetic kidney disease by antagonizing RANK/NF-κB signaling pathway and regulating autophagyObjective:(1)To investigate the role of OPG on DKD by animal experiments.(2)To confirm the possible mechanism of OPG acting on DKD.Methods:(1)4 db/m mice were selected as the normal control group.After successful modeling,20 db/db mice were randomly divided into DKD model group,OPG overexpression group,shOPG group,OPG NC group and shOPG NC group.(2)The level of serum OPG was detected by ELISA kit.Blood glucose,BUN and SCr were examined.(3)Renal tissue sections were stained with HE for general morphological analysis,PAS for mesangial matrix and basement membrane evaluation,and Masson for collagen deposition and interstitial lesions assessment,respectively.(4)The proportion of apoptotic cells was determined by the TUNEL assay.(5)The expression levels of RANK/NF-κB pathway related proteins(RANKL,TRAF6,P62 and P65)and autophagy related proteins(LC3B,Beclin-land Atg5)were detected by Real time PCR and Western blot.Results:(1)Compared with normal mice,the levels of OPG decreased significantly in each group of DKD mice.Compared with the DKD model group,the level of OPG in the overexpression OPG group was higher.(2)Compared with the DKD model group,the levels of BUN and SCr in the overexpression OPG group were significantly decreased,and the level of SCr in the shOPG group was significantly increased.(3)Compared with the DKD model group,the pathological changes of the kidney in the overexpression OPG group were significantly improved.For example,the mesangial cells were slightly proliferated,the increase and expansion of mesangial matrix decreased,the basement membrane thickening and the vacuolar degeneration of renal tubular epithelial cells were reversed to some extent,and the collagen fibers between glomerulus and tubules reduced significantly.(4)Compared with the DKD model group,the apoptosis of overexpression OPG group was decreased and the apoptosis of shOPG group was increased.(5)Compared with the DKD model group,the expressions of RANK/NF-κB pathway related proteins in overexpression OPG group were significantly decreased,while the expressions of autophagy related proteins were significantly increased,and all results in shOPG group were just the opposite.Conclusions:(1)Overexpressed OPG can protect kidneys and improve outcome in DKD.(2)OPG may protect the kidneys by antagonizing RANK/NF-κB signaling pathway and regulating autophagy.Part Three:The correlation between thyroid hormone levels and the kidney disease progression risk in patients with type 2 diabetesObjective:(1)To investigated the relationship between thyroid hormones and the risk of type 2 diabetic kidney disease(DKD)progression.(2)To obtain the cut-off points of thyroid hormones for assessing the high risk of DKD progression in different genders.Methods:(1)All subjects were patients with type 2 diabetes,routinely hospitalized in the Department of Endocrinology and Metabolism of the Third Hospital of Nanchang between February 2018 and November 2020.All subjects participated in the study voluntarily and signed written the informed consent form at admission.(2)Medical history collection,physical examination,and blood and urine samples testing were performed.(3)DKD was defined as eGFR<60 mL/min/1.73 m2 and/or ACR≥30 mg/g.Patients with or without DKD were grouped.The KDIGO classification was used to assess the risk of DKD progression(low,moderate,high or very high risks).Results:(1)Among the 452 subjects,188(41.6%)had diabetic nephropathy.(2)Compared with the patients without DKD,patients with DKD were a little younger,had lower eGFR,FT3 and FT4 levels;and higher BUN,SCr,UA,LDL-C,UACR,and TSH levels.(3)In subjects with DKD,the overall prevalence of thyroid dysfunction(any of hyperthyroidism or subclinical hyperthyroidism or hypothyroidism or subclinical hypothyroidism)was 22.9%,and the subclinical hypothyroidism was found in up to 19.7%of the population.The results were 17.5%and 9.5%in the non-DKD group,which were significantly different from DKD group.(4)FT3 levels varied with degrees of albuminuria,kidney damage,and KDIGO risk,and decreased with increasing severity.FT4 levels decreased with worsening CKD stages,and TSH levels increased with a higher risk of KDIGO categories.(5)There were negative correlations between FT3 and FT4 levels with both SCr and UACR,and positive correlations with eGFR.In contrast,there was a positive correlation between TSH levels and UACR,and a negative correlation with eGFR.(6)After adjustment,an increase in FT3 levels significantly reduced the risk of having DKD by 42%,and reduced the risk of DKD progression by 35%-50%.However,FT4 and TSH levels did not influence the risk of disease.(7)FT3 ≤4.30 pmol/L in men increased the high or very high risk of DKD progression by 2.66 times.FT3≤3.99 pmol/L in women increased the moderate risk of DKD progression by 1.37 times and increased the high or very high risk by 6.23 times.Conclusions:(1)Low FT3 level is an independent risk factor for having DKD and DKD progression.(2)Low FT3 levels,which are less than 4.30 pmol/L in men and less than 3.99 pmol/L in women,will greatly increase the risk of DKD progression.Part Four:Clinical application of fracture risk assessment tool(FRAX)in general populationObjective:(1)To analyze the characteristics of FRAX in general population.(2)To compare the differences of FRAX in urban and urban-rural fringe population.(3)To explore the effects of biochemical indicators of bone metabolism on FRAX.Methods:(1)Epidemiological investigation was conducted in Xihu District(urban area)and Qingshanhu district(urban-rural fringe area)of Nanchang City in June 2018.Residents aged 40 and over and signed informed consent were included.Physical examination,questionnaire data and oral glucose tolerance test were completed.(2)Blood samples of FBG,OGTT-2hBG,renal function and lipid levels were measured.Some biochemical indicators of bone metabolism(Ca,P,25-OH-VD,OC and β-CTX)were tested.(3)All subjects completed the BMD examinations of femoral neck and L1-L4 by DXA in the Third Hospital of Nanchang.In order to calculate the FRAX score,we clicked on the official website,and the results showed the probability of major osteoporotic fracture and hip fracture within the next 10 years.Results:(1)A total of 1051 subjects were included in this study,including 553 in urban area and 498 in urban-rural fringe area.The average BMD of L1-L4 was 0.78 ± 0.15g/cm2 and that of femoral neck was 0.77 ± 0.13g/cm2.The probability of major osteoporotic fracture and hip fracture within 10 years is 3.4(2.3-5.4)%and 0.6(0.3-1.5)%.(2)Compared with subjects in urban-rural fringe area,subjects in urban area had larger waist circumference,higher proportion of alcohol consumption,and higher levels of FBG and lipids.In contrast,there were fewer people suffering from rheumatoid arthritis and doing regular exercise,and the time of regular exercise was shorter.(3)Compared with urban population,the BMD of femoral neck in urban-rural fringe population was lower,FRAX scores were generally higher,Ca,P,β-CTX levels were lower,and 25-OH-VD level was higher.(4)FRAX scores of all subjects were mainly negatively correlated with BMD of L1-L4 and femoral neck,and with the level of Ca.There was an independent negative correlation between FRAX score and BMD of femoral neck in urban residents.In urban-rural fringe area,FRAX scores were mainly negatively correlated with BMD of L1-L4 and femoral neck.All FRAX scores showed an independent positive correlation with β-CTX levels.(5)In all subjects,or in the urban population or urban-rural fringe population,the increase of β-CTX levels significantly increased the risks of high probability of major osteoporotic fractures by 2.7 times,4.9 times and 31 times respective,and increased the risks of high probability of hip fracture by 3.5 times,6.4 times and 19.5 times in the next 10 years.Conclusions:(1)FRAX is a clinical tool worthy of promotion in general population.(2)The population in the urban-rural fringe has a high probability of fracture in the next 10 years.It is necessary to strengthen the early screening and intervention of osteoporosis and its fracture.(3)In addition to being related to BMD,increasing β-CTX level can significantly increase the risk of high probability of fracture,especially in urban-rural fringe population,suggesting that it needs to combine FRAX with BMD and biochemical indicators of bone metabolism for better predicting the risk of fracture.