前言：糖尿病肾病（diabetic nephropathy，DN）是糖尿病患者最常见的微血管并发症之一，在经济发达的国家和地区，DN已经成为终末期肾病的首要病因。系膜病变（系膜细胞病变和细胞外基质增生）是导致糖尿病肾小球硬化的始动因素和直接因素。VITAL研究（Lancet 2010; 376: 1543-1551）显示，在接受血管紧张素转换酶抑制剂或血管紧张素受体阻断剂（angiotensin converting enzyme inhibitor /angiotensin receptor blocker，ACEI/ARB）治疗的DN患者中，加用维生素D受体激动剂（帕立骨化醇2μg/日）可以显著降低尿蛋白，但维生素D降低DN患者尿蛋白的机制尚不完全清楚。1，25-二羟维生素D ₃ [1,25-Dihydroxyvitamin D₃，1,25(OH)₂ D₃]是体内维生素D的活性形式，已有的研究显示，它除了钙磷调节功能外，还具有调节细胞增殖、分化、凋亡等作用。我们推测1,25(OH)₂ D₃降低DN患者尿蛋白可能与抑制系膜细胞增殖有关。本研究分为两部分，第一部分：分析2型DN患者的临床、病理特征与肾脏预后的关系，探讨系膜病变与肾脏预后的相关性；第二部分：探讨1,25(OH)₂ D₃对高糖诱导的大鼠系膜细胞（rat mesangial cells ，RMCs）增殖的影响及其机制。

第一部分  2型糖尿病肾病患者的临床病理及其预后分析

目的：分析2型DN患者的临床、病理特征与肾脏预后的关系，探讨系膜病变与肾脏预后的相关性。

方法：回顾性研究2004年1月到2017年7月在中日友好医院行肾穿刺活检诊断为DN的126例患者，分析其临床基线指标、病理特征与肾脏预后的相关性。

结果：

本研究纳入126例DN患者，其中男性92例，女性34例，肾活检时平均年龄51.4±9.6岁，糖尿病病程9.3±6.0年，中位24h尿蛋白定量4.7g/d，血清肌酐153.9±55.4 μmol/L，估算的肾小球滤过率（estimated glomerular filtration rate，eGFR）57.9±22.3ml/(min·1.73m²)。

随着肾小球病变分期加重，糖尿病眼底病变加重、eGFR下降和贫血加重，Ⅲ期和Ⅳ期DN患者尤为明显。

单因素Cox回归分析显示24h尿蛋白定量、eGFR、血红蛋白、肾小球病变分期、间质纤维化及肾小管萎缩（interstitial fibrosis and tubular atrophy，IFTA）、间质炎症与肾脏预后有关。多因素Cox回归分析显示基线eGFR和肾小球病变分期是肾脏预后的独立危险因素。

DN患者肾脏局部血管紧张素AT1、AT2和MAS受体的表达与病理分期有一定相关性，在疾病早期有增加趋势，在疾病的末期表达下降。ARB治疗后，DN患者肾小球、肾小管间质和小动脉AT1受体表达下调(p < 0.05)，肾小管间质和小动脉MAS受体表达上调(p < 0.05)，AT2受体表达无明显变化。

结论：

多因素Cox回归分析显示基线eGFR和肾小球病变分期是肾脏预后的独立危险因素。

ARB治疗后，肾脏局部AT1受体表达下调，MAS受体表达上调。肾脏局部AT1受体在DN早期表达有增强趋势，在末期表达下降，提示ARB治疗应尽早开始。

第二部分  1,25(OH)₂ D₃拮抗高糖诱导的大鼠系膜细胞增殖效应的研究

目的：探讨1,25(OH)₂ D₃对高糖刺激的RMCs增殖的影响及其机制。

方法：Cell Counting Kit-8(CCK-8)方法检测细胞的增殖速率。流式细胞技术检测细胞周期变化。Western blot方法检测DDIT4/mTOR信号通路各个靶点VDR、DDIT4、 TSC1、TSC2、Rheb、 mTOR、4E-BP1和p70S6K蛋白的表达。

结果：

高糖促进RMCs增殖，1,25(OH)₂ D₃抑制高糖刺激的RMCs的增殖。

高糖组与低糖组和高渗组相比，VDR、DDIT4、TSC1、TSC2和4E-BP1 蛋白表达下调(p < 0.05)，Rheb、mTOR和p70S6K蛋白表达上调(p < 0.05)。高糖+VD组与高糖组相比，VDR、 DDIT4、TSC1、TSC2和4E-BP1蛋白表达上调(p < 0.05)，Rheb、mTOR和p70S6K蛋白表达下调(p < 0.05)。

RMCs分别转染空白质粒和DDIT4质粒。转染DDIT4质粒组与对照组相比，细胞增殖减弱。Western blot结果显示DDIT4、TSC1、TSC2和4E-BP1蛋白表达上调(p < 0.05)，Rheb、mTOR和p70S6K蛋白表达下调(p < 0.05)。

RMCs分别转染阴性对照siRNA和DDIT4 siRNA。DDIT4 siRNA组RMCs增殖加快，DDIT4 siRNA的作用与高糖类似，Western blot结果显示DDIT4、TSC1、TSC2和4E-BP1蛋白表达下调(p < 0.05)，Rheb、mTOR和p70S6K蛋白表达上调(p < 0.05)。1,25(OH)₂D₃刺激转染DDIT4 siRNA的RMCs，1,25(OH)₂D₃抑制细胞增殖的作用被DDIT4 siRNA所抑制。

结论：在体外实验中，1,25(OH)₂ D₃ 通过DDIT4/mTOR信号通路抑制高糖诱导的RMCs的增殖。本研究首次从正反两个方面证实，DDIT4是连接1,25(OH)₂ D₃ 与mTOR信号通路的重要因子。

全文结论：

DN系膜病变程度与肾脏预后密切相关。

肾脏局部AT1受体在DN早期表达有增强趋势，在末期表达下降，提示ARB治疗应尽早开始。

1,25(OH)₂ D₃通过DDIT4/mTOR信号通路抑制高糖刺激的RMCs的增殖。

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 nephropathy

Objective: 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:

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).

As glomerular lesions aggravated, diabetic retinopathy worsen, eGFR decreased, and anemia worsen, especially in patients with DN of class III and IV.

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.

The renal expression of AT1, 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:

Baseline eGFR and glomerular classification of DN were independent indicators for renal survival by multivariate Cox regression model.

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.

PartⅡ 1,25(OH)₂ D₃ inhibits the proliferation of rat mesangial cells induced by high glucose

Objective: 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:

Cell proliferation was promoted by high glucose and significantly reduced by 1,25(OH)₂ D₃.

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)₂ D₃ 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).

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.

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:

Mesangial lesions were closely related to renal prognosis of patients with type 2 DN.

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.

1,25(OH)₂D₃ inhibited the proliferation of RMCs induced by high glucose via the DDIT4/mTOR signaling pathway.