Effect Of Renal Lipid Deposition On Tubular Glucose Reabsorption And Its Molecular Mechanism

Part ?: Association of waist-to-hip ratio with urine glucose excretionBackground and objectives: The role of the kidney in glucose homeostasis has gained much more attention.The kidney contributes to glucose homeostasis largely through renal tubular glucose reabsorption.It has been reported that the capacity of renal tubular glucose reabsorption in particptants with diabetes is significantly higher than that of particptants with normal glucose tolerance.Increased tubular glucose reabsorption leads to an increase in the amount of glucose entering the blood circulation,thereby contributing to the progression of hyperglycemia.Therefore,promoting urine glucose excretion(UGE)through inhibition of renal glucose reabsorption has become an attractive approach for the treatment of diabetes.However,it is not clear that which factors that may contribute to increased renal glucose reabsorption.It is well known that obesity significantly increase the risk of type 2 diabetes.This study aimed to investigate the association of waist-to-hip ratio(WHR),a simple measure of abdominal obesity,with UGE determined in subjects without previous history of diabetes.Methods: A multistage,stratified sampling method was used to select a representative sample of individuals aged between 18 and 65 years in the general population from 6 cities in Jiangsu Province.A total of 7689 residents without previously diagnosed diabetes participated in this survey.A 75?g oral glucose tolerance test was used to diagnose diabetes.All urine samples were collected within 2 h of oral glucose loading to measure UGE.Information on age,sex,blood pressure,heart rate,height,weight,waist circumference,hip circumference was collected.Glycated hemoglobin(HbA1c),triglycerides(TG),total cholesterol(TC),high-density lipoprotein cholesterol(HDL-C),low-density lipoprotein cholesterol(LDL-C),creatinine,blood urea nitrogen(BUN)were detected.Multiple linear regression analysis and multivariate logistic regression analysis were performed to adjust the impact of potential confounding factors and explore the correlation between WHR and UGE.Results: 1.There were 7485 participants included in the final analysis after the exclusion of 204 participants whose data on HbA1 c or UGE were missing.597 individuals were newly diagnosed with diabetes mellitus(NDD),3645 individuals had prediabetes(PDM),and 3243 individuals had normal glucose tolerance(NGT).NDD group exhibited significantly higher UGE than NGT group and PDM group.A further subgroup analysis according to WHR was conducted.Participants were divided into high WHR(H-WHR)group and low WHR(L-WHR)group.Individuals with H-WHR exhibited significantly lower UGE compared to those with L-WHR,in either NGT group or PDM group.In NDD group,individuals with H-WHR also showed lower UGE than those with L-WHR,however,no statistical significance was observed.2.UGE was positively associated with age,heart rate,blood pressure,FPG,2h-PG,HbA1 c,TC,TG,LDL-C,BMI,and WHR(all p < 0.001),whereas negatively associated with HDL-C in the overall population.In addition,there was no significant correlation between UGE and creatinine.3.To identify the association of WHR with UGE,and eliminate the influence of potential confounders,a multiple linear regression analysis with UGE as a dependent variable was performed.WHR was negatively associated with UGE after adjustment for age,heart rate,genders,blood pressure,FPG,2h-PG,TG,TC,HDL-C,LDL-C,and BUN(? =-250.901,95% CI:-471.891 to-29.911,p = 0.026).However,there were no significant association between BMI and UGE after multivariable adjustment(p = 0.096).In addition,FPG,2h-PG,HbA1 c,and TG were still positively associated with UGE in this model.4.Furthermore,a binary logistic regression analysis was performed to identify the factors associated with the risk of high UGE.The result showed that H-WHR was significantly associated with a decreased odds ratio of high UGE in the multi-adjusted model(OR = 0.83,95% CI: 0.71-0.97,p = 0.018).Conclusion: Individuals with H-WHR were more likely to have low UGE.This study suggests that WHR,but not BMI,may be an important determinant of UGE,which means that the pattern of fat distribution,not overweight or obesity by itself,may be an important determinant of UGE.Part ?: Association of renal fat amount with renal threshold for glucoseBackground and objectives: Individuals with H-WHR were more likely to have low UGE,probably due to increased renal threshold for glucose(RTG).Increased RTG,which indicates renal glucose reabsorption is increased,leads to an increase in the amount of glucose entering the blood circulation,while a decrease in UGE.Accumulating evidences have demonstrated that RTG is increased in individuals with diabetes.However,little research has been designed to investigate factors that influence RTG.Visceral lipid deposition is the main driver of various metabolic disorders.Significant renal lipid deposition has been reported in hyperglycemic animals and humans.However,it is unclear that whether the renal lipid deposition may affect RTG.In addition,increased WHR reflects visceral fat accumulation,but it can not directly assess visceral fat amount.Therefore,this study aimed to evaluate renal fat amount using magnetic resonance imaging(MR)and further explore the correlation between renal fat amount and RTG.Methods: A total of 40 subjects were enrolled in the Department of Endocrinology,Zhongda Hospital Affiliated to Southeast University from December 2017 to April 2018.Baseline data were collected,including gender,age,blood pressure,heart rate,height,weight,waist circumference,hip circumference,etc.All participants underwent 3.0 T MR abdominal scan to assess renal fat amount.24-hour blood glucose was monitored and 24-hour urine smaples were collected to measure UGE.RTG was evaluated based on blood glucose and UGE.Simple correlation and partial correlation analyses were performed to investgate the correlation between renal fat amount and RTG.Results: 1.Seven subjects in this study were excluded.Five of them failed holding their breath during MR scanning and two withdrew from this study.Finally,a total of 33 subjects were included in this study.2.All subjects were devided into two groups when based on BMI.17 subjects with high BMI(high BMI,H-BMI)and 16 subjects with low BMI(low BMI,L-BMI).The renal fat fraction(RFF)of subjects with H-BMI was significantly higher than that of subjects with L-BMI(3.90% ± 1.09% vs 2.90% ± 1.06%,p < 0.05).In addition,subjects with diabetes had significantly higher RFF than that of non-diabetic subjects(4.18% ± 0.82% vs 2.25% ± 0.53%,p = 0.001).Moreover,RFF was significantly increased in subjects with high RTG(4.57% ± 0.97% vs 3.08% ± 1.07%,p = 0.001).3.The results of pearson correlation analysis showed that BMI and WHR were significantly and positively correlated with RFF.The correlation between WHR and RFF was the strongest(r = 0.537,p = 0.001).4.WHR was positively correlated with 24h-UGE(r = 0.371,p = 0.033),while BMI was not significantly associated with 24h-UGE(r = 0.235,p = 0.187).Furthermore,after adjusting for mean blood glucose,age,gender,diabetes status,estimated glomerular filtration rate(e GFR),and blood pressure,WHR was negatively associated with 24h-UGE(r =-0.405,p = 0.045).In addition,after adjusting for other confounding factors,there was a negative correlation trend between BMI and 24h-UGE,but there was no statistical significance.5.RFF was positively correlated with 24h-UGE(r = 0.396,p = 0.022).However,after adjusting for mean blood glucose,age,diabetes status,and e GFR,RFF was significantly and negatively correlated with 24h-UGE(r =-0.382,p = 0.041).6.BMI,WHR and RFF were positively correlated with RTG,and the correlation between RFF and RTG was the strongest.After adjusting for mean blood glucose,age,gender,diabetes status,e GFR and blood pressure,BMI and WHR were no longer significantly associated with RTG.However,RFF was still positively correlated with RTG(r = 0.432,p = 0.031).Moreover,after adjusting for mean blood glucose,age,gender,diabetes status,e GFR,blood pressure,BMI,and WHR,RFF was still positively correlated with RTG(r = 0.433,p = 0.039).Conclusion: RFF was negatively correlated with UGE and positively correlated with RTG,suggesting that renal lipid deposition may be an important factor for increased RTG.Renal lipid deposition may promote renal tubular glucose reabsorption,thereby resulting in reduced urine glucose excretion.Part ?: Effect of lipid deposition on renal SGLT2 expressionBackground and objectives: The role of lipotoxicity in the pathogenesis of diabetes mellitus by inducing insulin resistance and islet function damage has been well confirmed.However,it is still unclear whether lipotoxicity will participate in the pathogenesis of diabetes mellitus through other approaches.In recent years,much more attention has been paid to the role of kidney in glucose homeostasis.It has been well known that the kidney contributes to glucose homeostasis largely through glucose reabsorption.Sodium-glucose cotransporter 2(SGLT2)is the key protein that determines renal glucose reabsorption and the dominant molecule that determines RTG.The second part found RFF was significantly and positively correlated with RTG,suggesting that the renal lipid deposition might be an important factor for RTG.However,it is unclear that whether renal lipid deposition will affect SGLT2 expression.To date,there is no study focuse on the association of renal lipid deposition with SGLT2 expression.Therefore,this study aimed to clarify the effect of renal lipid deposition on SGLT2 expression through animal experiments,and furtherly explore the association of signaling pathway related to lipid synthesis liver X receptor(LXR)/sterol regulatory element binding protein 1c(SREBP1c)with SGLT2 expression levels.Methods: SD rats were randomly divided into two groups,one group was given a normal diet(control,CON)and another group was given a high fat diet(HFD).The obese rats were constructed by feeding with high fat diet continuously for 16 weeks.Successful models were defined as the weight of HFD group was more than 20% of that of CON group.Both groups were given an intraperitoneal glucose tolerance test(IPGTT)and an intraperitoneal insulin tolerance test(IPITT).24-hour urine smaples were collected using metabolic cage to quantitatively detect UGE.Blood lipids,glucose,and creatinine were measured.Oil red O staining and free fatty acid(FFA)quantitative assessment were used to assess the renal fat amount.Immunofluorescence staining,real-time fluorescence quantitative polymerase chain reaction(q PCR),and wstern blot were conducted to determine renal SGLT2,LXR,SREBP1 c expression levels.Results: 1.The body weight and kidney weight of rats in HFD group were significantly higher than those in CON group(p < 0.05).Blood glucose was monitored and there was no significant difference in the mean blood glucose between the two groups(p = 0.358).2.The results of IPGTT and IPITT showed that the rats in the HFD group did not meet the diagnostic criteria for diabetes,but already had abnormal glucose tolerance and insulin resistance.3.The RTG of rats in HFD group was 6.04 ± 0.23mmol/L,higher than that in CON group 5.88 ± 0.14mmol/L,but the difference was not statistically significant(p = 0.33).4.The results of oil red O staining showed that significant renal tubular lipid deposition was observed in the HFD group,but no lipid deposition was found in the kidney of rats in the CON group.Quantitative detection of renal cortical FFA showed that the FFA levels in HFD group were significantly higher than that in CON group(P < 0.001).5.The results of immunofluorescence staining,q PCR and WB exhibited that the expression of SGLT2 in HFD group was significantly higher than that in CON group.In addition,the expression of lipid synthesis related proteins LXR? and SREBP1 c in HFD group were markedly higher,while there was no significant difference in LXR? expression between the two groups.Pearson correlation analysis showed that the FFA levels,LXR? and SREBP1 c protein expression levels were positively correlated with SGLT2 protein expression levels.Conclusion: Compared with CON group,the m RNA and protein expression of SGLT2 in HFD group was significantly upregulated,which may be related to renal lipid deposition caused by activation of LXR?/SREBP1 c signaling pathway.Part ?: Effect of free fatty acids on SGLT2 expression in tubular epithelial cellsBackground and objectives: The levels of free fatty acid(FFA)in the blood of individuals with obesity or type 2 diabetetes are significantly higher than that in the normal population.Palmitic acid(PA)is the most abundant saturated FFA in the circulation.It is well known that when FFA exceeds the storage capacity of adipose tissue,it will deposit in non-adipose tissue,thereby causing damage to the tissue and cells,which is called lipotoxicity.Obvious lipid deposition was observed in the kidney of obese rats.In addition,SGLT2 expression was significantly up-regulated in the obese rats.The result of the third part suggested that the renal lipid deposition might be an important factor for the overexpression of SGLT2.However,the result of the third part still could not identify that improving lipid deposition could alleviate the up-regulation of SGLT2.Therefore,this study aimed to furtherly verify the effect of lipid deposition on SGLT2 expression by cell experiments,and confirm whether SGLT2 expression will be alleviated by improving celluar lipid deposition through inhibiting signaling pathway related to lipid synthesis LXR?/SREBP1 c,and finally reveal lipid deposition is an important factor for overexpression of SGLT2.Methods: Human proximal tubular epithelial cells(HK2 cells)were cultured in different groups,PA treatment group and normal control group.Oil red O staining was used to evaluate the intracellular lipid deposition.The expression levels of SGLT2,LXR and SREBP1 c were determined by immunofluorescence staining,q PCR and WB.Moreover,the glucose uptake of each group was measured.In addition,in order to inhibit signaling pathway related to lipid synthesis,HK2 cells were transfected with si RNA-LXR? and si RNA-SREBP1 c,then the cells were treated with PA again.Intracellular lipid deposition,SGLT2 expression levels,and glucose uptake were evaluated again.Results: 1.After treatment with 200 ?mol/L PA for 24 hours,obevious lipid deposition in HK2 cells was observed.2.The result of immunofluorescence staining showed that normal HK2 cells expressed SGLT2 protein.In addition,the average fluorescence intensity of PA-treated cells was significantly higher than that of normal control group.Moreover,the results of q PCR and WB also showed that the expression of SGLT2 in PA treatment group was significantly up-regulated compared to normal control group.Furthermore,compared with the normal control group,the PA treatment group exhibited a significant increase in glucose uptake(P < 0.05).3.The expressions of LXR? and SREBP1c m RNA and protein in PA treatment group were significantly higher than those in the normal control group.However,there was no significant difference in LXR? expression levels between the two groups.5.After transfection with si RNA-LXR? and si RNA-SREBP1,the intracellular lipid deposition was significantly improved compared with PA treatment group.Moreover,SGLT2 expression was obviously reduced.In addition,compared with the PA treatment group,glucose uptake was significantly attenuated in HK2 cells transfected with si RNA-LXR? and si RNA-SREBP1.Conclusion: Activation of LXR?/SREBP1 c signaling pathway promotes intracellular lipid deposition,which in turn leads to upregulation of SGLT2 in renal tubular epithelial cells,ultimately affecting glucose uptake.Lipid deposition is an important factor for overexpression of SGLT2.