| Background:Diabetic nephropathy(DN)is one of the most common microvascular complications of diabetes and the leading cause of end-stage kidney disease in developed countries.Mesangial lesions(proliferation of mesangial cells and extracellular matrix expansion)have been considered as contributing factors to the initial pathophysiologic mechanisms involved in glomerulosclerosis.VITAL study(Lancet 2010;376:1543-1551)showed addition of vitamin D receptor activation(2 μg/day paricalcitol)to RAAS inhibition safely lowers residual albuminuria in patients with DN,however,the mechanism was not fully clear.1,25-Dihydroxyvitamin D3[1,25(OH)2 D3],the hormonal form of vitamin D,it has new functions independent of calcium and phosphorus homeostasis,such as regulating cell proliferation,differentiation,apoptosis,etc.We assume that 1,25(OH)2D3 lowers residual albuminuria in patients with DN may be related to inhibiting the proliferation of mesangial cells.This study was divided into two parts.The first part was to analyze the clinicopathologic features and renal prognosis of patients with type 2 DN,and to explore the relationship between mesangial lesions and renal prognosis.The second part was to explore the effect of 1,25(OH)2D3 on the proliferation of rat mesangial cells(RMCs)induced by high glucose and its mechanism.Part Ⅰ Clinicopathologic features and prognosis of patients with type 2 diabetic nephropathyObjective:To analyze the clinicopathologic features and renal prognosis of patients with type 2 DN,and to explore the relationship between mesangial lesions and renal prognosis.Methods:126 patients diagnosed as type 2 DN by renal biopsy in China-Japan Friendship Hospital between January 2004 and July 2017 were enrolled in this study.The clinicopathologic features,and the correlation between baseline data and renal prognosis were analyzed.Results:1.This study enrolled 126 patients,including 92 males and 34 females.The average age at renal biopsy was 51.4±9.6 years,the duration of diabetes was 9.3±6.0 years,the median 24-hour urinary protein was 4.7 g/d,and serum creatinine was 153.9±55.4 μmol/L,estimated glomerular filtration rate(eGFR)57.9±22.3 ml/(min· 1.73m2).2.As glomerular lesions aggravated,diabetic retinopathy worsen,eGFR decreased,and anemia worsen,especially in patients with DN of class Ⅲ and Ⅳ.3.24-hour urinary protein,eGFR,hemoglobin,glomerular classification of DN,interstitial fibrosis and tubular atrophy(IFTA),and interstitial inflammation were related to renal prognosis by univariate Cox regression model.Baseline eGFR and glomerular classification of DN were independent indicators for renal survival by multivariate Cox regression model.4.The renal expression of ATI,AT2,and MAS receptors had a certain correlation with glomerular classification of DN.The expression of AT1,AT2,and MAS receptors tended to increase during the early stages of DN,but they decreased during the late stage.When DN patients were treated with ARB,the expression of AT1 receptors was downregulated in glomeruli,tubulointerstitium,and vascular(p<0.05),the expression of MAS receptors was upregulated in tubulointerstitium and vascular(p<0.05),but there was no difference in AT2 receptor expression.Conclusions:1.Baseline eGFR and glomerular classification of DN were independent indicators for renal survival by multivariate Cox regression model.2.When DN patients were treated with ARB,the renal expression of AT1 receptors was downregulated,the expression of MAS receptors was upregulated.The renal expression of AT1 receptor tended to increase during the early stages of DN,but it decreased during the late stage,suggesting that patients with DN should use ARB as soon as possible.Part Ⅱ 1,25(OH)2 D3 inhibits the proliferation of rat mesangial cells induced by high glucoseObjective:To explore the effect of 1,25(OH)2D3 on the proliferation of RMCs induced by high glucose and its mechanism.Methods:The cell proliferation rate was measured using cell counting kit-8 assay(CCK-8).Cell cycle duration was examined using flow cytometry.Protein expression of DDIT4/mTOR signaling pathway(VDR,DDIT4,TSC1,TSC2,Rheb,mTOR,4E-BP1 and p70S6K)was assayed by western blot.Results:1.Cell proliferation was promoted by high glucose and significantly reduced by 1,25(OH)2D3.2.The protein levels of VDR,DDIT4,TSC1,TSC2,and 4E-BP1 were significantly downregulated(p<0.05)and those of Rheb,mTOR,and p70S6K were significantly upregulated(p<0.05)in HG group vs.LG group and Man group.Incubation of RMCs with 1,25(OH)2D3 for 48 h increased VDR expression(p<0.05),restored the expression of DDIT4,TSC1,TSC2 and 4E-BP1(p<0.05),and blocked the aberrant upregulation of Rheb,mTOR and p70S6K(p<0.05).3.RMCs were transiently transfected with blank vector or DDIT4 vector.The short-term overexpression of DDIT4 suppressed RMCs proliferation.The protein levels of DDIT4,TSC1,TSC2,and 4E-BP1 were significantly upregulated(p<0.05),Rheb,mTOR,and p70S6K were significantly downregulated(p<0.05)in cells transfected with the DDIT4 vector.4.RMCs were transfected with Negative Control siRNA and DDIT4 siRNA.DDIT4 siRNA promoted cell proliferation similar to high glucose.The expression of DDIT4,TSC1,TSC2,and 4E-BP1 were significantly downregulated(p<0.05)and Rheb,mTOR,and p70S6K proteins were significantly upregulated(p<0.05).siRNA knockdown of DDIT4 completely abrogated antiproliferative responses to 1,25(OH)2D3.Conclusions:In vitro,1,25(OH)2D3 inhibited the proliferation of RMCs induced by high glucose via the DDIT4/mTOR signaling pathway.For the first time,it was confirmed that DDIT4 is an important factor connecting the 1,25(OH)2 D3 and mTOR signaling pathways from both positive and negative sides.The conclusions of this study:1.Mesangial lesions were closely related to renal prognosis of patients with type 2 DN.2.The renal expression of AT1 receptor tended to increase during the early stages of DN,but it decreased during the late stage,suggesting that patients with DN should use ARB as soon as possible.3.1,25(OH)2D3 inhibited the proliferation of RMCs induced by high glucose via the DDIT4/mTOR signaling pathway. |
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