1.Correlation Between Follicle Stimulating Hormone Level And Renal Dysfunction In Post-menopausal Women 2.Correlation Between Serum Free Fatty Acid Levels And Incident Diabetes In A Cohort Study

IntroductionChronic kidney disease(CKD)is a major public health problem,with a global prevalence estimated at 5-10%.The burden of CKD-associated diseases is alarmingly high,which is primarily due to cardiovascular morbidity and mortality.Kidney function is influenced by sex hormones.Earlier researching findings suggested that female sex hormones were overall protective against the progression of kidney disease;however,a meta-analysis of 11 studies revealed a more rapid decline in kidney function in women(who were mostly post-menopausal)compared to men.Moreover,the renoprotection associated with female sex hormones appears to diminish after menopause,and epidemiologic studies have reported a substantial increase in CKD incidence in women after menopause.A recent study in the United States reported that women-mostly post-menopausal—accounted for 42%of patients on dialysis.These findings suggest that the changes in sex hormone levels associated with menopause have adverse effects on kidney function.Oestrogen attenuates glomerulosclerosis and tubulointerstitial fibrosis;thus,the decline in oestrogen level after menopause can promote the progression of CKD.The occurrence of CKD was found to be lower in women who received oral hormone replacement therapy(HRT)than in those who did not receive the treatment.However,oestrogen supplementation did not completely reverse the decline in renal function in post-menopausal women,indicating that factors other than oestrogen withdrawal contribute to the observed decline in renal function following menopause.Menopause is characterised by the absence of menstrual cycles as a result of ovarian failure and decreased circulating oestrogen concentrations along with a compensatory increase in circulating follicle-stimulating hormone(FSH).FSH acts on non-gonadal tissues through the FSH receptor.We previously reported that FSH affects hepatic cholesterol biosynthesis,hepatic gluconeogenesis,and osteoarthritis.In the present study,we investigated the association between FSH levels and renal function in women in the context of menopause;we also examined whether FSH is an independent risk factor for renal dysfunction in post-menopausal women,and the influence of age on this association.Objectives:In this study,pre-menopausal,peri-menopausal and post-menopausal women were selected from the epidemiological survey population of our research group.By comparing the sex hormone levels and renal function indices differences among pre-menopausal,peri-menopausal and post-menopausal women,the relationship between menopause and renal function decline was preliminarily explored.Post-menopausal women were divided according to serum FSH level quartiles to explore the change trend of renal function with the increase of FSH quartile level.Adjusting for confounding variables,the independent risk of FSH in post-menopausal women with renal dysfunction was assessed.The modification effect of age on the relationship between FSH and renal function was further investigated.Materials and methods1.Study design and participantsThis cross-sectional study was conducted in Ningyang County,Shandong Province,China in 2014 as part of the Risk Evaluation of cAncers in Chinese diabeTic Individuals:A lONgitudinal(REACTION)trial at Shanghai Jiao Tong University School of Medicine(clinical trial number:NCT01506869).The study was approved by Ruijin Hospital Ethics Committee of Shanghai JiaoTong University School of Medicine.Informed consent was obtained from each participant after a detailed explanation of the purpose and nature of all procedures used.The population was composed of two parts:6060 people were followed up from 2011 and 2862 people were newly added in 2014.Of the 8922 participants in the REACTION trial,3285 eligible women were included in the present study who met the following criteria:1)available key data such as age,body mass index(BMI),renal function indices,sex hormone levels,serum lipid profiles,menstrual history,hypertension,diabetes,etc.;2)no condition affecting the natural state of menopause and renal function such as premature ovarian failure,bilateral ovariectomy,uterectomy,pregnancy,lactation,chronic nephritis,nephrotic syndrome,nephrectomy,and malignant tumours;and 3)no use of medications affecting the natural state of menopause and renal function including oestrogens,androgens,progesterone,glucocorticoids,thiazide diuretics,methoxyflurane,tetracycline,penicillin,and sulphonamides.The women were assigned to one of three groups according to menopausal status—ie,pre-menopause(n=624),peri-menopause(n=121),and post-menopause(n=2540).2.Data collectionAll investigators completed a standardised training program to minimise inter-rater variability.For pre-menopausal women,venipuncture was scheduled on days 2-5 of a regular menstrual cycle to exclude periodic differences in sex hormone levels;for peri-and post-menopausal women,venipuncture was performed at random times.All blood samples were collected between 8:00 a.m.and 10:00 a.m.after overnight fasting and immediately preserved at-80℃ until use.Blood glucose level was measured within 2 h.Female sex hormone levels were evaluated by chemiluminescence(Cobas E601;Roche,Basel,Switzerland).The sensitivity for FSH detection was 0.100 mIU/ml,and the range of measurement was 0.100-200.0 mIU/ml;for E2,the sensitivity and range of measurement was 5 pg/ml and 5-3000 pg/ml respectively.Intra-and inter-assay coefficients of variation were always<5%for FSH and E2.Serum lipid profiles,plasma glucose levels,and indices of hepatic and renal functions were examined using a Beckman AU5800 chemistry analyser(Beckman Coulter,Tokyo,Japan).Blood pressure was measured three times at 3-min intervals for each participant using an electronic sphygmomanometer(HEM-7117;Omron,Kyoto,Japan),and the mean value was calculated.Height(cm)and weight(kg)were recorded and used to calculate BMI(kg/m2).3.Definitions of study outcomesMenopausal status was determined based on responses to a self-report questionnaire regarding menstrual history or amenorrhea.Pre-menopause was defined as the presence of menses within the past 3 months.Peri-menopause was defined as the presence of menses within the past 3 months with menstrual irregularity in the year preceding the questionnaire,or 3-11 months of amenorrhea.The former close to the early peri-menopausal period,the latter close to the late peri-menopausal period.It has been reported that estrogen did not significantly decline in early peri-menopausal women due to the compensatory effect of increased FSH.In order to present the gradient of estrogen decline,we selected late peri-menopausal women to be included in the study(i.e.,3-11 months of amenorrhea).Post-menopause was defined as the cessation of menstruation for a minimum of 12 months.Renal dysfunction was defined as declined estimated glomerular filtration rate(eGFR<90 ml/min/1.73 m2)or CKD(eGFR<60 ml/min/1.73 m2).Dyslipidaemia was defined as follows:1)high total cholesterol(≥ 6.22 mmol/l);2)high triglyceride(≥1.70 mmol/1);3)high low-density lipoprotein cholesterol(≥ 4.14 mmol/l);4)high free fatty acids(≥ 0.9 mmol/1);and 5)low high-density lipoprotein cholesterol<1.30 mmol/l).Hypertension and diabetes were diagnosed based on self-reported previous diagnosis,or were defined as systolic blood pressure≥130 mmHg or diastolic blood pressure≥ 85 mmHg for hypertension and fasting plasma glucose≥7.0 mmol/l or post-prandial 2-h plasma glucose≥11.1 mmol/l for diabetes.Never smoking or drinking was assigned a value of 0;otherwise,it was assigned a value of 1.4.Statistical analysisStatistical analyses were performed using SPSS v24.0 for Windows(IBM Corp,Armonk,NY,USA).Continuous variables with normal and skewed distributions are presented as mean±standard deviation and median with interquartile range,respectively.Categorical variables are presented as numbers and percentages.Comparisons of various indices among the three menopausal states or among FSH quartiles in post-menopausal women were performed by one-way analysis of variance(ANOVA)for continuous variables with normal distribution;with the Kruskal-Wallis test for continuous variables with skewed distribution;and with the chi-squared test for categorical variables.ANOVA and Cochran-Armitage chi-squared test were used to assess the trends in continuous and categorical variables,respectively,in the transitions across menopausal states or increasing FSH quartiles in post-menopausal women.A linear regression model was used to evaluate the relationship between FSH levels and eGFR levels in post-menopausal women.A multivariate logistic regression model was used to evaluate the association of FSH quartiles with the prevalence of renal dysfunction in post-menopausal women with adjustments for age,oestradiol(E2),BMI,dyslipidaemia,hypertension,diabetes,smoking,and drinking.The outcomes of the logistic regression analysis are presented as regression coefficient(B),adjusted odds ratio(OR),and 95%confidence interval(CI).All calculated p values were two-sided,and p<0.05 was considered statistically significant.Results1.Sex hormone levels and renal function indices in pre-,peri-,and post-menopausal womenThe 3285 women included in the study were divided into pre-menopause(n=624),peri-menopause(n=121),and post-menopause(n=2540)groups.With the change in menopausal status from pre-menopause to peri-and post-menopause,median serum E2 level decreased sharply from 66.75 to 19.12 to 5.58 pg/ml,while median serum FSH level increased from 8.65 to 56.49 to 71.50 mIU/ml.Age,which is an irreversible risk factor for renal dysfunction,increased significantly across the three groups from 43.06 to 51.36 to 60.94 years(p for trend<0.001).The prevalence of decreased eGFR increased from pre-menopause to peri-and post-menopause(6.1%to 14.9%to 49.0%;p for trend<0.001),with a corresponding increase in prevalence of CKD(p for trend=0.001).Additionally,in post-menopausal women,mean serum creatinine(Scr)increased from 57.47 to 64.55 μmol/1 and mean eGFR level decreased from 108.27 to 88.73 ml/min/1.73 m2(both p<0.001 vs pre-menopausal women)2.Renal function declines with increasing FSH level in post-menopausal womenWe divided post-menopausal women into quartiles according to serum FSH level(mIU/ml)as follows:quartile 1:FSH≥56.78 mIU/ml,quartile 2:56.78<FSH≥71.61 mIU/ml,quartile 3:71.61<FSH≥88.35 mIU/ml,and quartile 4:FSH>88.35 mIU/ml.With increasing FSH quartile,mean Scr level increased from 62.98 to 66.55 μmol/l(p for trend<0.001)whereas eGFR level decreased from 90.70 to 87.03 ml/min/1.73 m2(p for trend<0.001).The post hoc test showed that mean Scr or eGFR levels between any two FSH quartiles differed significantly(p<0.05),except between quartiles 2 and 3(p=0.408 and 0.388,respectively).Similarly,the prevalence of declined eGFR increased from 41.4%to 53.9%(p for trend<0.001)while that of CKD increased from 0.3%to 3.3%(p for trend<0.001)with increasing FSH quartiles.On the other hand,age did not show any trend across FSH quartiles(p for trend=0.316)and E2 levels were extremely low in all quartiles.Linear regression analysis showed that FSH levels were negatively associated with eGFR levels in post-menopausal women even after adjustment for potential confounders,such as age,years since menopause,LH,E2,BMI,lipid profiles(including TC,TG,LDL-C,LDL-C,HDL-C,FFA),diabetes,hypertension,smoking,drinking(Beta=-0.128 in model 1;Beta=-0.125 in model 2;Beta=-0.172 in model 3;all p values<0.001).These results indicate that FSH has negative relationship with renal function in post-menopausal women.3.FSH is an independent risk factor for renal dysfunction in post-menopausal womenTo investigate whether FSH contributes to renal dysfunction in post-menopausal women,we conducted a multivariate logistic stepwise regression analysis.After adjusting for potential confounders including age,years since menopause,LH,E2,BMI,dyslipidaemia,diabetes,hypertension,smoking,and drinking,the ORs of declined eGFR and CKD were two-fold(OR=2.187,95%CI:1.635-2.924)and 10-fold(OR=10.088,95%CI:2.279-44.650)higher,respectively,in the highest FSH quartile compared to the lowest(p<0.05).4.Age influences the association between FSH and renal dysfunction in post-menopausal womenWe stratified the post-menopausal women into two equal-sized groups(n=1270 each)according to median age(<61 and>61 years)and conducted a multivariate logistic stepwise regression analysis for each group.After adjusting for age,years since menopause,LH,E2,BMI,dyslipidaemia,diabetes,hypertension,smoking,and drinking,the ORs of declined eGFR increased across FSH quartiles in both age groups;moreover,they were higher for each FSH quartile in the older group(OR=1.461 for quartile 2,OR=2.486 for quartile 3,and OR=2.530 for quartile 4 vs quartile 1)than for the corresponding quartile in the younger group(OR=0.925 for quartile 2,OR=1.498 for quartile 3,and OR=1.755 for quartile 4 vs quartile 1).Conclusion:1.The renal function of postmenopausal women decreased with the increase of FSH level2.High FSH is an independent risk factor for renal dysfunction in post-menopausal women3.Aging might aggravate the adverse effects of FSH on renal function in post-menopausal womenBackground:Diabetes is a major medical problem in China and worldwide due to its increasing incidence and prevalence.Hundreds of million individuals suffer from diabetes(type-1 or type-2 diabetes mellitus)and its various cardiovascular complications,causing a heavy health burden worldwide.In 2017,the International Diabetes Federation revealed that approximately 425 million individuals had diabetes worldwide and this number is predicted to increase to 629 million by 2045.In China,the prevalence of diabetes and prediabetes in adults were estimated at 11.6%and 50.1%,respectively.These figures equate to approximately 113.9 and 493.4 million Chinese adults with diabetes and prediabetes,respectively.Therefore,it is necessary to understand the pathogenesis of diabetes and identify factors with causal roles in the etiology of diabetes.There is an extensive body of basic science and animal research that has explored the role of free fatty acid(FFA)in glucose intolerance or the development of diabetes.Chronically increased serum FFA or sustained hyper-free fatty acidemia induce hepatic and muscle insulin resistance and impair insulin secretion referred to as lipotoxicity,which are considered the two main pathological mechanisms of diabetes.However,relatively few longitudinal epidemiologic studies have investigated the associations between serum FFA levels and incident diabetesPrevious cross-sectional studies have examined the relationship between fasting circulating FFA concentrations and diabetes and draw a common conclusion that individuals with diabetes have higher circulating FFA concentrations compared with normal individuals.However,the results of longitudinal cohort studies assessing whether baseline levels of circulating FFA can predict the risk of developing diabetes remain controversial.The majority of prospective epidemiological studies reported that high FFA levels predict the development of diabetes after adjustment for potential confounders such as obesity.By contrast,another study reported no relationship between baseline FFA levels and the development of Type 2 diabetes,even showing that baseline FFA levels were slightly lower in those who progressed to diabetes than with those who did not.The differences in study populations may at least partially explain the variation in results observed across previous studies.However,the effect of FFA on glucose metabolism is a chronic and long-term process.To the best of our knowledge,no investigation has evaluated the associations between longitudinal changes in serum FFA or mean serum FFA levels and incident diabetes.Therefore,in this study,we investigated the associations between baseline serum FFA levels and incident diabetes.Further,we explored the risk associated with the longitudinal statuses of serum FFA across the incident diabetes follow-up.Objective:1.To evaluate the correlation between baseline FFA level and the incidence of diabetes at the end of follow-up,and to study the predictive value of high baseline FFA level on the incidence of diabetes.2.To evaluate the correlation between the longitudinal status of FFA and the incidence of diabetes,including the longitudinal change of FFA level from baseline to the end of follow-up,and the mean level.Materials and methods1.Study design and participantsThis retrospective cohort study was derived from a noninterventional epidemiological investigation titled Risk Evaluation of cAncers in Chinese diabeTic Individuals:A longitudinal(REACTION)study in Shanghai Jiao Tong University School of Medicine(clinical trial number:NCT01506869).The REACTION study enrolled 259,657 Chinese people(≥ 40 years of age)from 25 communities in China’s mainland between 2011 and 2012 with follow-ups planned at 3-,5-,and 10-years.The data included in the present study are from study participants from Ningyang County,Shandong Province.The 3-year follow-up visits were conducted from 2011 to 2014.Overall,7,068 subjects participated in the baseline survey.Individuals who met the following criteria were included in this retrospective cohort study:(1)individuals with non-diabetes(normal or prediabetes)at baseline;(2)blood samples were examined at baseline and at follow-up to acquire available serum indices,including serum lipid profiles such as serum triglyceride(TG),total cholesterol(TC),low-density lipoprotein cholesterol(LDL-C),high density lipoprotein cholesterol(HDL-C),FFA,fasting blood glucose(FPG),2-hour postprandial blood glucose(2hPG),and glycosylated hemoglobin(HbA1c)levels;(3)no missing information of key covariates including age,body mass index(BMI),waist circumference(WC),family history of diabetes,and history of smoking and drinking;(4)no use of medications affecting glucose and lipid metabolism,including metformin,insulin,statins and fibrates three months before recruitment and during the follow-up period;and(5)no disease affecting glucose and lipid metabolism including malignant tumors,severe hepatic and renal insufficiency,and thyroid dysfunction three months before recruitment and during the follow-up.Ultimately,2905 individuals were enrolled in this study.This study was approved by the Ruijin Hospital Ethics Committee of Shanghai JiaoTong University School of Medicine.Informed consent was obtained from each participant after receiving a detailed explanation of the purpose and nature of all procedures used.This study was conducted in accordance with the Declaration of Helsinki.2.Definition of groupings and outcomesSerum FFA levels at baseline or the end of follow-up were classified as:normal(FFA<0.9 mmol/L)or high(FFA≥ 0.9 mmol/L)according to the normal reference ranges(0.1-0.9 mmol/L)in Department of Laboratory Medicine,Shandong Provincial Hospital.Longitudinal changes in FFA levels from the baseline to the end of follow-up were categorized as:from normal to normal;from normal to high;from high to normal;and from high to high.Additionally,mean serum FFA and ΔFFA levels from the baseline to the end of follow-up were calculated using the following formulas:mean FFA level=(FFA concentration at baseline+FFA concentration at end of follow-up)/2,and △FFA level=FFA concentration at end of follow-up-FFA concentration at baselineThe glucose metabolic status of participants at baseline and at the 3-year follow-up included normal,prediabetes,and diabetes,defined based on the ADA 2017 criteria.Criteria for the diagnosis of diabetes were:FPG>126 mg/dL(7.0 mmol/L)or 2hPG≥ 200 mg/dL(11.1 mmol/L)after an oral glucose tolerance test(OGTT)or HbA1c≥ 6.5%(48 mmol/mol).Criteria for the diagnosis of prediabetes were FPG,100 mg/dL(5.6 mmol/L)to 125 mg/dL(6.9 mmol/L)(IFG)or 2hPG after an OGTT,140 mg/dL(7.8 mmol/L)to 199 mg/dL(11.0 mmol/L)(IGT)or HbA1c,5.7%-6.4%(39-47 mmol/mol).The OGTT should be performed as described by the WHO,using a glucose load containing the equivalent of 75 g anhydrous glucose dissolved in water.3.Data collectionAll investigators completed a standardized training program to minimize inter-rater variability.All blood samples were collected between 8:00 and 10:00 AM after overnight fasting and were immediately preserved at-80℃ until required for analysis.Blood glucose levels were examined within 2 h.Serum lipid profiles including FFA,glucose metabolic indices including FPG and 2hPG,HbAlc,and indices of hepatic and renal function were examined using a Beckman AU5800 chemistry analyzer system(Beckman Coulter,Tokyo,Japan).For laboratory investigation,at least 1 ml of serum was taken to run above lab tests and the response rate of participants towards blood sampling was more than 90%.For each of the above parameters,intra-and inter-assay coefficients of variation were always below 5%.Blood pressure was measured three times at 3-min intervals for each participant between 8:00 AM and 10:00 AM in sitting position using an electronic sphygmomanometer(HEM-7117;Omron,Kyoto,Japan)and the mean value was calculated.WC was measured in centimeters.Weight and height were measured in kilograms and centimeters,respectively.BMI was calculated by dividing the weight(kilograms)by the square of the height(square meters).Family history of diabetes,and history of smoking and drinking were based on the results of self-reported questionnaires.4.Statistical analysisStatistical analyses were performed using IBM SPSS Statistics for Windows,version 25.0.The means ± standard deviation and medians(interquartile range)were used to describe continuous variables with normal and skewed distributions,respectively.Categorical variables are presented as numbers(percentages).Continuous variables with normal distribution were compared using a t-test or one-way analysis of variance(ANOVA).Continuous variables with skewed distribution were compared by Mann-Whitney U test.Categorical variables were compared by chi-square test.With increasing FFA quartiles,ANOVA trend tests and Cochran-Armitage chi-squared tests for trend were used to assess the trends of continuous and categorical variables,respectively.A multivariate logistic stepwise regression model adjusted for age,sex,family history of diabetes,BMI,WC,FPG,TC,TG,LDL,HDL,and smoking and drinking levels was used to assess the risk correlation between FFA and incident diabetes.Logistic regression analysis outcomes are presented as regression coefficients(B),adjusted odds ratio(OR)values,and 95%confidence intervals(CIs).All calculated p values were two-sided,and p<0.05 was considered statistically significant.Results1.Baseline characteristics of the study population according to FFA levelsThe basic clinical characteristics of the study population according to FFA levels are summarized in Table 1.Compared with individuals with normal FFA levels,the mean age and indices of glucose metabolism such as FPG and 2hPG were significantly increased in individuals with high FFA levels,while the mean HbAlc between the two groups did not statistically differ(p>0.05).The prevalence of prediabetes was significantly higher among individuals with high FFA levels than among individuals with normal FFA levels(85.7%vs.80.8%,p<0.05).The proportion of family history of diabetes was significantly greater among individuals with normal FFA levels than among participants with high FFA levels(2.5%vs.1.3%,p<0.05).Additionally,individuals with high FFA levels had higher TC,TG,and HDL-C levels.For obesity indices,individuals with high FFA levels had lower BMI and WC,while there was no statistically significant difference(both p>0.05).Finally,renal function in individuals with high FFA levels was slightly lower.Among the baseline population,a total of 290 of 2,905 participants developed diabetes by the 3-year follow-up.2.Associations of FFA levels at baseline with various indices of glucose metabolism and risk of incident diabetes at end of follow-upThe pooled population were divided by baseline FFA levels.Compared with individuals with normal baseline FFA levels,individuals with high baseline FFA levels had higher FPG,2hPG,HbAlc levels and a higher proportion of diabetes at the end of follow-up.However,the mean FPG and HbA1c in groups with normal and high baseline FFA levels did not statistically differ(both p>0.05).The pooled population was then divided into quartiles based on baseline FFA levels as follows:quartilel:FFA ≤ 0.48,quartile2:0.48<FFA ≤ 0.63,quartile3:0.63<FFA ≤ 0.81,and quartile4:FFA>0.81(mmol/L).With increasing FFA quartiles,the mean levels of FPG,2hPG,and HbAlc,and the proportion of diabetes at the end of follow-up increased in a stepwise manner.The trends of increased FPG and HbAlc were not statistically significant.To further evaluate whether baseline FFA levels were independently associated with incident diabetes,we conducted multivariate logistic stepwise regression analyses.After adjusting for vital confounders such as age,sex,BMI,WC,family history of diabetes,FPG,TG,TC,LDL-C,HDL-C,and smoking and drinking levels,all adjusted ORs for incident diabetes showed no statistical significance irrespective of how baseline FFA levels were modeled(normal/high FFA,FFA quartiles,or continuous variable).3.Longitudinal increase and sustained high level status of serum FFA levels are associated with higher risk of incident diabetesLongitudinal changes of FFA levels from the baseline to the end of follow-up were categorized into four types:from normal to normal;from normal to high;from high to normal;and from high to high.Furthermore,levels of mean serum FFA and△FFA from baseline to the end of follow-up were calculated.After adjusting for confounding factors such as age,sex,BMI,WC,family history of diabetes,FPG,TG,TC,LDL-C,HDL-C,and smoking and drinking levels,individuals with the serum FFA levels from normal to high(OR=2.956,95%CI:2.089-4.184)and from high to high(OR=3.343,95%CI:2.300-4.857)were associated with higher risk for incident diabetes than were individuals with the serum FFA levels from normal to normal.Individuals with △FFA≥ 0 mmol/L had a higher risk of incident diabetes(OR=1.762,95%CI:1.373-2.262,p<0.001)compared with the individuals with △FFA<0 mmol/L.When individuals were divided by mean FFA exposure,the risk for incident diabetes(OR=2.120,95%CI:1.620-2.775)increased approximately twofold among individuals with mean FFA≥ 0.9 mmol/L compared with that among individuals with mean FFA<0.9 mmol/LConclusion:In conclusion,the longitudinal status of serum FFA levels,including chronic increases and sustained high levels,is more closely associated with high risk of incident diabetes than is high baseline FFA levels.Thus,a reduction in elevated circulating FFA should be an important target when attempting to postpone or prevent the occurrence and development of diabetes.More emphatically,the longitudinal long-term control of circulating FFA level,such as weight loss and some pharmacological interventions,are helpful to prevent the occurrence of diabetes.