Abstract
目的
尿维生素D结合蛋白(urine vitamin D-binding protein，uVDBP)的排泄可能参与2型糖尿病(Type 2 diabetic mellitus，T2DM)患者早期肾损害的发生和发展，本研究旨在探讨uVDBP在T2DM患者中的检测意义及其与肾小管的关系，为T2DM肾损害的早期诊断提供新的方向。
方法
纳入符合入组标准的105例T2DM患者(患者组)和同期募集健康体检者30例(正常对照组)，收集所有入组人员的一般资料及血、尿液生物化学指标，并采用ELISA法检测uVDBP、尿人肾损伤因子-1(urine kidney injury molecule 1，uKIM-1)、尿中性粒细胞明胶酶相关脂质运载蛋白(urine neutrophil gelatinase-associated lipocalin，uNGAL)、尿视黄醇结合蛋白(urine retinol-binding protein，uRBP)水平，再经尿肌酐(creatinine，Cr)校正为uVDBP/Cr、uKIM-1/Cr、uNGAL/Cr、uRBP/Cr。采用Pearson和Spearman相关性检验分析uVDBP/Cr与尿白蛋白肌酐比值(urine albumin-to-creatinine ratio，UACR)、肾小球滤过率估计值(estimated glomerular filtration rate，eGFR)及肾小管损伤标志物之间的相关性；采用多重线性回归和受试者操作特征曲线分析uVDBP/Cr与UACR和eGFR的相关性。
结果
与正常对照组相比，患者组的uVDBP/Cr升高(P<0.05)，且与UACR呈正相关(r=0.774，P<0.01)；与eGFR呈负相关(r=-0.397，P<0.01)。两组之间的uKIM-1/Cr、uNGAL/Cr、uRBP/Cr水平差异均有统计学意义(均P<0.01)；uVDBP/Cr与uKIM-1/Cr(r=0.752，P<0.01)、uNGAL/Cr(r=0.644，P<0.01)、uRBP/Cr(r=0.812，P<0.01)均呈正相关。uVDBP/Cr对T2DM早期肾损害患者评估的灵敏度为90.0%，特异度为82.9%；eGFR对T2DM早期肾损害患者评估的灵敏度为75.0%，特异度为72.6%。
结论
uVDBP/Cr可以作为早期评估T2DM患者肾损害的生物学标志物。
Keywords: 糖尿病, 维生素D结合蛋白, 早期肾损害
Abstract
Objective
The excretion of urinary vitamin D-binding protein (uVDBP) is related to the occurrence and development of early-stage renal damage in patients with Type 2 diabetes (T2DM). This study aims to explore the significance of detecting uVDBP in T2DM patients and its relationship with renal tubules, and to provide a new direction for the early diagnosis of T2DM renal damage.
Methods
A total of 105 patients with T2DM, who met the inclusion criteria, were included as a patient group, and recruited 30 individuals as a normal control group. The general information and blood and urine biochemical indicators of all subjects were collected; the levels of uVDBP, and a marker of tubular injury [urine kidney injury molecule 1 (uKIM-1), urine neutrophil gelatinase-associated lipocalin (uNGAL) and urine retinol-binding protein (uRBP)] were detected by enzyme-linked immunosorbent assay. The results were corrected by urinary creatinine (Cr) to uVDBP/Cr, uKIM-1/Cr, uNGAL/Cr and uRBP/Cr. The Pearson’s and Spearman’s correlation tests were used to analyze the correlation between uVDBP/Cr and urine albumin-to-creatinine ratio (UACR), estimated glomerular filtration rate (eGFR) and markers of tubular injury, and multivariate linear regression and receiver operating characteristic curve were used to analyze the correlation between uVDBP/Cr and UACR or eGFR.
Results
Compared with the normal control group, the uVDBP/Cr level in the patient group was increased (P<0.05), and which was positively correlated with UACR (r=0.774, P<0.01), and negatively correlated with eGFR (r=-0.397, P<0.01). There were differences in the levels of uKIM-1/Cr, uNGAL/Cr, and uRBP/Cr between the 2 groups (all P<0.01). The uVDBP/Cr was positively correlated with uKIM-1/Cr (r=0.752, P<0.01), uNGAL/Cr (r=0.644, P<0.01) and uRBP/Cr (r=0.812, P<0.01). The sensitivity was 90.0% and the specificity was 82.9% (UACR>30 mg/g) for evaluation of uVDBP/Cr on T2DM patients with early-stage renal damage, while the sensitivity was 75.0% and the specificity was 72.6% for evaluation of eGFR on T2DM patients with early-stage renal damage.
Conclusion
The uVDBP/Cr can be used as a biomarker in early-stage renal damage in T2DM patients.
Keywords: diabetic mellitus, urine vitamin D binding protein, early-stage renal damage
2型糖尿病(Type 2 diabetic mellitus，T2DM)是一种以血糖浓度升高为特征的代谢性紊乱疾病，也是世界上患病率增长最快的慢性疾病之一[1]。糖尿病肾病(diabetic kidney disease，DKD)特指由糖尿病所致的慢性肾病(chronic kidney disease，CKD)，存在着高致残和致死风险。大数据研究[2]认为DKD可能将替代原发性肾小球病成为终末期肾病(end-stage renal disease，ESRD)的首要病因。
肾小球是DKD损伤的主要靶点，目前尿白蛋白肌酐比值(urine albumin-to-creatinine ratio，UACR)和肾小球滤过率估计值(estimated glomerular filtration rate，eGFR)被公认为评价DKD严重程度的标志物。随着其在临床的广泛应用，有临床研究[3-4]发现在某些患者中UACR和eGFR并不是早期DKD的敏感标志物，最为重要的是，以上两个指标都主要是因肾小球损伤而发生的变化。不仅如此，DKD各阶段人群的尿肾损伤分子-1(urine kidney injury molecule 1，uKIM-1)[5]、尿视黄醇结合蛋白(urine retinol-binding protein，uRBP)[6]、尿中性粒细胞明胶酶相关脂质运载蛋白(urine neutrophil gelatinase-associated lipocalin，uNGAL)[7]等标志物的排泄也是增加的，且排泄量与肾功能减退的程度相平行。多项研究[8-9]表明肾小管间质病变与DKD患者的肾功能存在密切关系，这可能也是影响患者预后的关键因素之一。
维生素D结合蛋白(vitamin D-binding protein，VDBP)是于1959年在人血浆中发现的新型多功能球蛋白，其特异的重吸收机制可导致正常人尿维生素D结合蛋白(urine vitamin D-binding protein，uVDBP)排泄量极低。首次发现uVDBP排泄量增加与肾损伤严重程度相关的研究[10]是在CKD人群中开展的。多项临床研究[11-12]也表明uVDBP在T2DM人群尤其在DKD人群中排泄增加，但这些研究结论大多提及的是uVDBP的排泄可能会引起T2DM患者的维生素D缺乏，或可能与T2DM患者早期肾损伤有关，未明确VDBP与肾小球功能和肾小管功能损伤的关系。因此本研究探讨T2DM患者中uVDBP与uKIM-1、uNGAL和uRBP等肾小管损伤标志物的相关性，以明确uVDBP检测对T2DM早期肾损伤患者的诊断意义。
1. 对象及方法
1.1. 对象
收集495例2018年8月至2019年12月在中南大学湘雅三医院(以下简称我院)肾内科和内分泌科的住院患者；同期在我院健康管理中心募集年龄、性别相匹配的30名健康志愿者。入选标准：符合美国糖尿病协会[13]、中华医学会糖尿病学分会[14]2017年T2DM诊断标准的成年人。排除标准：eGFR<30 mL/(min·1.73 m-2)的患者；患有急性感染[15]、严重创伤[16]、自身免疫性疾病[17]、肝功能不全[18]、造影剂[19]等影响VDBP水平的疾病及肾毒性药物使用史[20]的患者；妊娠(或哺乳期)妇女；肿瘤、糖尿病酮症酸中毒、血液系统疾病或其他内分泌及代谢性疾病的患者。根据入选标准共计纳入105例患者。本研究获得中南大学湘雅三医院伦理委员会的批准(审批号：I-22303)，所有受试者均知情同意。
1.2. 方法
记录所有入组人员的姓名、性别、年龄、住院日期、既往病史等。收集常规的身高、体重、血常规、肝肾功能、血脂、血压、尿沉渣、血肌酐、空腹静脉血糖、糖化血红蛋白、血清白蛋白、尿微量白蛋白、尿肌酐、泌尿系彩色B超及其他基本影像学资料等。根据2017年美国糖尿病协会[13]、中华医学会糖尿病学分会[14]发布的指南中尿液白蛋白排泄异常的定义，将患者按UACR分为如下3组：1)Nomo组。UACR值<30 mg/g的患者，共计35例。2)Micro组。UACR值的范围为30~300 mg/g的患者，共计34例。3)Macro组。UACR值>300 mg/g的患者，共计36例。根据2007年美国肾脏病基金会肾脏病预后质量倡议工作组指南[21]的CKD定义，即按简化肾脏病饮食改良研究公式计算eGFR值，并对入组患者进行如下分组：1)CKD1期组。eGFR值≥90 mL/(min·1.73 m-2)，共计37例。2)CKD2期组。eGFR值的范围为60~89 mL/(min·1.73 m-2)患者，共计36例。3)CKD3期组。eGFR值的范围为30~59 mL/(min·1.73 m-2)患者，共计32例。上述两种分组中，正常对照组(NC组)均为健康志愿者，且均为30例。
在清晨收集符合标准的受试者的中段尿6 mL，分装于1.5 mL的无菌EP管中，在30 min内离心(1 000 g，15 min、4 ℃)血浆标本及尿液标本，离心结束后用无菌移液管吸取上清液置于无菌的1.5 mL EP管中，储存于-80 ℃冰箱，采用ELISA法检测uVDBP、uKIM-1、uNGAL、uRBP的水平，在反应终止后5 min内采用酶标仪在450 nm波长处依序测量各孔的光密度(OD)值，将OD值转换为以ng/mL为单位的浓度。为避免尿量的影响，uVDBP、uKIM-1、uNGAL、uRBP的水平根据尿肌酐(creatinine，Cr)进行归一化，以uVDBP/Cr、uKIM-1/Cr、uNGAL/Cr、uRBP/Cr表示。
根据《中国2型糖尿病防治指南(2017年版)》[14]，以UACR>30 mg/g或eGFR<60 mL/(min·1.73 m-2)为T2DM患者肾功能损害的诊断标准。
1.3. 统计学处理
采用SPSS 19.0统计学软件进行数据分析。计量资料进行正态性检验，若符合正态分布，用均数±标准差( ±s)表示，2组比较采用方差分析法；不符合正态分布，用中位数和四分位间距[M(P 25，P 75)]表示，2组比较采用Mann-Whitney U秩和检验，多组间比较采用 Kruskal-Wallis检验。使用Pearson和Spearman相关性检验分析uVDBP与肾小管损伤指标的相关性，采用多元线性回归方程分析uVDBP与UACR及eGFR的相关性，受试者操作特征曲线(receiver operator characteristic，ROC)探讨uVDBP和肾小管损伤指标对早期DKD肾功能损害的诊断效能，以P<0.05为差异有统计学意义。
2. 结 果
2.1. 一般临床资料及生物化学指标的改变
2.1.1. 按UACR分组的结果
Nomo组、Micro组、Macro组T2DM患者的年龄、性别、高密度脂蛋白及总胆固醇与NC组相比，差异均无统计学意义(均P>0.05)；3组患者的BMI、空腹血糖、三酰甘油、收缩压、舒张压、uVDBP/Cr及eGFR均较NC组升高(均P<0.05)；3组患者的白蛋白、eGFR均较NC组降低(均P<0.05)；低密度脂蛋白质只有Nomo组较NC组降低(P<0.05)；Macro组血红蛋白及血Cr均较NC组升高(均P<0.05)；Macro组的eGFR较NC组降低(P<0.05，表1)。
表1.
各组临床资料和生化指标的比较(按UACR分组)
Table 1 Comparison of clinical data and biochemical indicators in each group (grouping based on UACR)
| 组别 | n | 男/女 | 年龄/岁 | BMI/(kg·m-2) | 空腹血糖/(mmol·L-1) | 低密度脂蛋白/(mmol·L-1) | 高密度脂蛋白/(mmol·L-1) |
|---|---|---|---|---|---|---|---|
| NC组 | 30 | 20/10 | 50.87±12.30 | 22.10±2.75 | 5.17(4.82~5.40) | 2.77±0.76 | 1.20±0.43 |
| Nomo组 | 35 | 24/11 | 55.26±11.76 | 25.95±4.22* | 9.00(7.50~11.45)* | 2.15±0.88* | 1.14±0.25 |
| Micro组 | 34 | 24/10 | 54.29±11.56 | 25.49±6.35* | 8.80(7.25~11.325)* | 2.45±1.00 | 1.10±0.25 |
| Macro组 | 36 | 25/11 | 56.33±10.27 | 25.27±3.31* | 7.53(5.63~10.70)* | 2.43±1.08 | 1.21±0.28 |
| 组别 | 总胆固醇/(mmol·L-1) |
三酰甘油/ (mmol·L-1) |
收缩压/mmHg | 舒张压/mmHg | uVDBP/Cr /(ng·mg-1) |
|---|---|---|---|---|---|
| NC组 | 4.70 | 1.12 | 114.50 | 67.50 | 3.12 |
| Nomo组 | 4.74(3.70~5.51) | 1.83(1.30~2.95)* | 130.00(121.50~138.50)* | 80.00(75.50~87.50)* | 13.29(7.43~17.78)* |
| Micro组 | 4.89(4.07~5.24) | 2.13(1.34~2.52)* | 140.00(124.25~149.75)* | 84.00(76.25~88.75)* | 28.88(19.48~48.98)*† |
| Macro组 | 4.7(3.65~5.57) | 1.50(1.09~2.67) | 153.50(136.25~165.25) *†‡ | 85.00(78.00~90.50)*† | 116.23(71.64~155.73)*†‡ |
| 组别 | 糖化血红蛋白/% | 白蛋白/(g·L-1) | 血红蛋白/(g·L-1) | 血肌酐/(μmol·L-1) |
eGFR/ [mL/(min·1.73 m-2)] |
|---|---|---|---|---|---|
| NC组 | — | 47.78 | 143.50 | 62.00 | 105.50 |
| Nomo组 | 9.56±2.39 | 40.80(38.90~42.50)* | 140.00(131.50~145.00) | 69.00(57.00~84.50) | 89.00(72.00~116.00)* |
| Micro组 | 9.56±2.35 | 42.40(39.73~43.45)* | 137.00(128.25~147.75) | 73.50(59.25~97.75)* | 82.50(67.25~122.00)* |
| Macro组 | 8.78±2.10 | 32.85(28.48~36.98)*†‡ | 117.00(94.50~128.75)*†‡ | 113.00(84.00~158.25)*†‡ | 57.50(33.00~128.75)*†‡ |
1 mmHg=0.133 kPa。与正常对照组相比，*P<0.05；与Nomo组相比，†P<0.05；与Micro组相比，‡P<0.05。UACR：尿白蛋白肌酐比值；BMI：体重指数；uVDBP：尿维生素D结合蛋白；Cr：尿肌酐；eGFR：肾小球滤过率估计值。
2.1.2. 按eGFR分组的结果
与NC组相比，CKD1期组、CKD2期组、CKD3期组T2DM患者的性别、高密度脂蛋白及总胆固醇差异均无统计学意义(均P>0.05)；BMI、空腹血糖、三酰甘油、收缩压、舒张压、白蛋白、UACR、uVDBP/Cr均升高(均P<0.05)；CKD2期组、CKD3期组年龄较NC组及CKD1期组升高(均P<0.05)；CKD1期组低密度脂蛋白较NC组降低(P<0.05)；CKD3期组血红蛋白较NC组降低(P<0.05)；CKD2期组及CKD3期组血肌酐均较NC组升高(均P<0.05)；CKD3期组uVDBP/Cr较CKD1期、2期组均升高(均P<0.05，表2)。
表2.
各组临床资料和生化指标比较(按eGFR分组)
Table 2 Comparison of clinical data and biochemical indicators in each group ( grouping based on the eGFR)
| 组别 | n | 男/女 | 年龄/岁 | BMI/(kg·m-2) |
空腹血糖/ (mmol·L-1) |
低密度脂蛋白/(mmol·L-1) | 高密度脂蛋白/(mmol·L-1) |
|---|---|---|---|---|---|---|---|
| NC组 | 30 | 20/10 | 50.87±12.30 | 22.10±2.75 | 5.17(4.82~5.40) | 2.77±0.76 | 1.20±0.43 |
| CKD1期 | 37 | 26/11 | 49.19±8.62 | 25.14±6.12* | 8.60(7.50~12.30)* | 2.20±0.91* | 1.11±0.27 |
| CKD2期 | 36 | 21/15 | 60.19±9.51*† | 26.10±4.35* | 9.05(7.13~11.78)* | 2.42±0.98 | 1.14±0.23 |
| CKD3期 | 32 | 20/12 | 56.91±12.32*† | 25.46±3.13* | 8.03(5.86~9.90)* | 2.43±1.11 | 1.21±0.29 |
| 组别 | 总胆固醇/(mmol·L-1) |
三酰甘油/ (mmol·L-1) |
收缩压/mmHg | 舒张压/mmHg | uVDBP/Cr/(ng·mg -1) |
|---|---|---|---|---|---|
| NC组 | 4.65±0.88 | 1.12(0.74~1.45) | 114.53(107.25~122.25) | 69.00±7.23 | 3.12(1.82~5.64) |
| CKD1期 | 4.68±1.22 | 2.04(1.09~2.96)* | 132.00(116.00~149.00)* | 83.84±9.55* | 18.91(13.29~41.13)* |
| CKD2期 | 5.11±2.01 | 1.85(1.20~2.46)* | 132.00(122.25~148.00)* | 81.53±9.51* | 30.27(14.10~71.32)* |
| CKD3期 | 4.83±1.66 | 1.85(1.14~2.87)* | 149.50(134~161)*†‡ | 82.78±9.82*† | 78.84(46.03~137.67)*†‡ |
| 组别 | 糖化血红蛋白/% | 白蛋白/(g·L-1) | 血红蛋白/(g·L-1) | 血肌酐/(μmol·L-1) | UACR/(mg·g -1) |
|---|---|---|---|---|---|
| NC组 | — | 47.78(45.15~49.30) | 143.50(133.00~57.00) | 62.00(52.25~0.75) | 2.77(1.99~3.57) |
| CKD1期 | 9.95±2.28 | 41.00(39.70~43.60)* | 137(129.00~47.00) | 59.00(52.00~7.00) | 32.39(9.60~112.35)* |
| CKD2期 | 9.08±2.16 | 39.80(36.33~41.50) *† | 137.5(129.00~47.00) | 83.50(72.75~8.00) *† | 52.73(12.11~34.40)* |
| CKD3期 | 8.78±2.33 | 33.00(25.58~9.15) *†‡ | 118.00(94.5~29.50) *†‡ | 121.50(104.00~73.25) *†‡ | 634.14(131.50~305.65) *†‡ |
1 mmHg=0.133 kPa。与NC组相比，*P<0.05；与CDK1期组相比，†P<0.05；与CKD2期组相比，‡P<0.05。UACR：尿白蛋白肌酐比值；BMI：体重指数；uVDBP：尿维生素D结合蛋白；Cr：尿肌酐；eGFR：肾小球滤过率估计值。
2.2. uVDBP/Cr与UACR及eGFR相关性分析
uVDBP/Cr与UACR呈正相关(r=0.774，P<0.01)，将uVDBP/Cr及UACR对数转换后见图1。
图1.
uVDBP/Cr与UACR相关分析对数散点图
Figure 1 Logarithmic scatter plot of correlation analysis between uVDBP/Cr level and UACR uNGAL: Urine neutrophil gelatinase-associated lipocalin; Cr: Creatinine; UACR: Urine albumin-to-creatinine ratio.
uVDBP/Cr与eGFR呈负相关(r=-0.397，P<0.01)，将uVDBP/Cr对数转换后见图2。
图2.
uVDBP/Cr与eGFR相关分析对数散点图
Figure 2 Logarithmic scatter plot of correlation analysis between uVDBP/Cr and eGFR uNGAL: Urine neutrophil gelatinase-associated lipocalin; Cr: Creatinine; eGFR: Estimated glomerular filtration rate.
Spearman相关性分析提示uVDBP/Cr与UACR存在显著正相关，以UACR为因变量进行对数转换后，以BMI、空腹血糖、低密度脂蛋白、三酰甘油、收缩压、舒张压、uVDBP/Cr、白蛋白、血红蛋白、血肌酐为自变量进行多元线性逐步回归，结果发现血Cr、uVDBP/Cr及收缩压可以被纳入方程，从而获得uVDBP表达的回归方程：Y=-1.80+1.042(uVDBP/Cr)+0.008(收缩压)-0.093(白蛋白)+0.043(舒张压)，决定系数R 2=0.649，提示uVDBP/Cr与UACR呈独立相关(表3)。
表3.
UACR的多元线性逐步回归
Table 3 Multiple linear stepwise regression of UACR
| 变量 | B | β | t | P |
|---|---|---|---|---|
| uVDBP/Cr | 1.042 | 0.123 | 8.452 | 0.000 |
| 收缩压 | 0.008 | 0.008 | 2.193 | 0.310 |
| 白蛋白 | -0.093 | 0.024 | -3.912 | 0.000 |
| 舒张压 | 0.043 | 0.017 | 2.540 | 0.013 |
UACR：尿白蛋白肌酐比值；uVDBP：尿维生素D结合蛋白；Cr：尿肌酐；UACR：尿白蛋白肌酐比值。
Spearman相关性分析提示：uVDBP/Cr与eGFR存在显著负相关，以eGFR为因变量，以BMI、空腹血糖、低密度脂蛋白、三酰甘油、收缩压、舒张压、uVDBP/Cr进行多元线性逐步回归，结果发现收缩压、uVDBP/Cr、空腹血糖可以逐步被纳入方程，从而获得eGFR表达的回归方程：Y=158.563-0.454(收缩压)-7.523(uVDBP/Cr)+1.811(空腹血糖)，决定系数R 2=0.280，提示uVDBP/Cr与eGFR独立相关(表4)。
表4.
eGFR的多元线性逐步回归
Table 4 Multiple linear stepwise regression of eGFR
| 变量 | B | β | t | P |
|---|---|---|---|---|
| 收缩压 | -0.454 | -0.296 | -3.383 | 0.001 |
| uVDBP/Cr | -7.523 | -0.309 | -3.575 | 0.001 |
| 空腹血糖 | 1.811 | 0.194 | 2.273 | 0.025 |
uVDBP：尿维生素D结合蛋白；Cr：尿肌酐；eGFR：肾小球滤过率估计值。
2.3. uVDBP/Cr与肾小管损伤标志物表达水平相关性分析
将105名T2DM患者按UACR分组，所有数值均用尿Cr校正，结果显示Macro组uKIM-1/Cr、uNGAL/Cr、uRBP/Cr水平较Micro组及Nomo组均升高(均P<0.01)，Micro组uRBP/Cr水平较Nomo组升高(P<0.01，表5)。
表5.
T2DM患者尿液肾小管标志物水平比较(按UACR分组)
Table 5 Comparison of urinary tubular marker levels in patients with T2DM (grouping based on the DUACR)
| 组别 | n | uKIM-1/Cr | uNGAL/Cr | uRBP/Cr |
|---|---|---|---|---|
| Nomo组 | 35 | 0.85 | 2.71 | 0.15 |
| Micro组 | 34 | 1.10 | 3.34 | 1.19** |
| Macro组 | 36 | 2.98**†† | 16.21**†† | 16.28**†† |
与Nomo组相比，**P<0.01；与Micro组相比，††P<0.01。UACR：尿白蛋白肌酐比值；uKIM-1：尿人肾损伤因子；Cr：尿肌酐；uNGAL：尿中性粒细胞明胶酶相关脂质运载蛋白；uRBP/Cr：尿视黄醇结合蛋白。
将105名T2DM患者按eGFR分组，所有数值均用尿Cr校正，结果显示CKD3期组uKIM-1/Cr、uNGAL/Cr、uRBP/Cr水平较CKD2期组及CKD1期组均升高(均P<0.01)，CKD2期组uRBP/Cr水平较CKD1期组升高(P<0.01，表6)。
表6.
T2DM患者尿液肾小管标志物水平比较(按eGFR分组)
Table 6 Comparison of urinary tubular marker levels in patients with T2DM (grouping based on the eGFR)
| 组别 | n | uKIM-1/Cr | uNGAL/Cr | uRBP/Cr |
|---|---|---|---|---|
| CKD1组 | 37 | 1.10 | 2.71 | 0.43 |
| CKD2组 | 36 | 1.16 | 3.56 | 4.05** |
| CKD3组 | 32 | 2.21**†† | 14.75**†† | 9.90**†† |
与CKD1组相比，**P<0.01；与CKD2组相比，††P<0.01。eGFR：肾小球滤过率估计值；uKIM-1：尿人肾损伤因子；Cr：尿肌酐；uNGAL：尿中性粒细胞明胶酶相关脂质运载蛋白；uRBP/Cr：尿视黄醇结合蛋白。
将uVDBP/Cr与uKIM-1/Cr、uNAGL/Cr及uRBP/Cr进行相关性分析，结果显示T2DM患者uVDBP/Cr水平与uKIM-1/Cr水平呈明显正相关(r=0.752，P<0.01)，将数据对数转换后见图3A；T2DM患者uVDBP/Cr水平与uNGAL/Cr水平呈明显正相关(r=0.644，P<0.01)，将数据对数转换后见图3B；T2DM患者uVDBP/Cr水平与uRBP/Cr水平呈明显正相关(r=0.812，P<0.01)，将数据对数转换后见图3C。
图3.
uVDBP/Cr与肾小管损伤标志物相关分析对数散点图
Figure 3 Logarithmic scatter plot of correlation analysis between uVDBP/Cr and renal tubular injury markers
A: Logarithmic scatter plot of correlation analysis between uVDBP/Cr expression level and uKIM-1/Cr level; B: Logarithmic scatter plot of correlation analysis between uVDBP/Cr expression level and uNGAL/Cr level; C: Logarithmic scatter plot of correlation analysis between uVDBP/Cr expression level and uRBP/Cr level. uVDBP: Urine vitamin D-binding protein; Cr: Creatinine; uNGAL: Urine neutrophil gelatinase-associated lipocalin; uRBP: Urine retinol-binding protein.
2.4. uVDBP和eGFR诊断糖尿病早期肾损伤的ROC曲线分析
以UACR>30 mg/g为T2DM患者肾功能损害的诊断标准，获得的结果如下：uVDBP/Cr曲线下面积为0.900，最佳临界值为18.849 0 ng/mg(灵敏度为90.0%，特异度为82.9%)；uKIM-1/Cr曲线下面积为0.703，最佳临界值为1.208 9 ng/mg(灵敏度为68.6%，特异度为85.7%)；uNGAL/Cr曲线下面积为0.737，最佳临界值为6.802 1 ng/mg(灵敏度为58.6%，特异度为88.6%)；uRBP/Cr曲线下面积为0.972，最佳临界值为0.534 7 ng/mg(灵敏度为92.1%，特异度为94.3%)(图4)。
图4.
T2DM患者UACR的ROC曲线分析
Figure 4 ROC curve analysis of UACR in patients with T2DM
uVDBP: Urine vitamin D-binding protein; Cr: Creatinine; uKIM-1: Urine kidney injury molecule 1; uNGAL: Urine neutrophil gelatinase-associated lipocalin; uRBP: Urine retinol-binding protein; ROC: Receiver operator characteristic; T2DM: Type 2 diabetes; UACR: Urine albumin-to-creatinine ratio.
以eGFR<60 mL/(min·1.73 m-2)为T2DM患者肾功能损害的诊断标准[14]，获得的结果如下：uVDBP/Cr曲线下面积为0.749，最佳临界值为47.821 ng/mg(灵敏度为75.0%，特异度为72.6%)；uKIM-1/Cr曲线下面积为0.719，最佳临界值为1.735 ng/mg(灵敏度为68.8%，特异度为74.0%)；uNGAL/Cr曲线下面积为0.839，最佳临界值为6.465 ng/mg(灵敏度为81.3%，特异度为72.6%)；uRBP/Cr曲线下面积为0.769，最佳临界值为6.970 ng/mg(灵敏度为60.7%，特异度为75.7%)(图5)。
图5.
T2DM患者eGFR的ROC曲线分析
Figure 5 ROC curve analysis of eGFR in patients with T2DM
uVDBP: Urine vitamin D-binding protein; Cr: Creatinine; uKIM-1: Urine kidney injury molecule 1; uNGAL: Urine neutrophil gelatinase-associated lipocalin; uRBP: Urine retinol-binding protein; ROC: Receiver operator characteristic; T2DM: Type 2 diabetes; eGFR: Estimated glomerular filtration rate.
3. 讨 论
UACR与eGFR是用来判断T2DM患者肾损害的重要生物标志物，但其临床诊断效能可能仍存在一定的局限性：其一，高血压、肥胖等病情、药物的使用均可以影响蛋白尿而导致该指标的特异性较低；其二，肾小管损伤可提前亦可独立于肾小球病变。虽然DKD的相关机制尚不完全明确，但早期诊断和及时治疗对逆转或延缓患者的肾功能损伤是极为关键的，因此致力于探寻筛选能力强且有临床意义的新型无创生物标志物是必要的。
本研究发现T2DM患者的uVDVP/Cr水平显著上升，这一点与Shoukry等[12]的研究结论一致。在有关血清VDBP的研究中，Fawzy等[22]发现T2DM患者血清VDBP较健康人群是升高的，但补充说明可能是炎症反应状态或代偿机制所致，王媛等[23]的研究显示DKD患者的血清VDBP水平与uVDBP水平呈正相关，但此研究也未排除感染的影响。而Nyomba等[24]在糖尿病老鼠模型中发现血清VDBP水平是下降的，临床研究[25]中1型糖尿病患者的血清VDBP也呈下降趋势。鉴于血清VDBP水平变化机制复杂，有关结论并不一致，为规避影响，本研究严格把握了入选指征，将患有可能影响血清VDBP水平变化的急性感染[15]、严重创伤[16]、自身免疫性疾病[17]、肝功能不全[18]等疾病的患者均不纳入实验，单独探讨uVDBP对于T2DM患者肾功能损伤的预测意义。
本研究发现uVDBP/Cr与UACR存在显著正相关，uVDBP/Cr与eGFR呈负相关，且均为独立相关，uVDBP/Cr可能是肾功能损伤的生物学标志物，其中UACR的升高既与肾小球功能的改变相关，也能提示肾小管功能损伤[26]，因此进一步分析uVDBP/Cr与肾小管功能的相关性。KIM-1在健康人群的肾组织中少量表达，这提示存在肾小管损伤[27]。NGAL是脂蛋白家族成员之一，在肾单位损伤后释放，可作为慢性肾小管间质纤维化的早期标志物[28]。RBP为一种 21 kD(1 D=1 u)蛋白质，由肾小球滤过后运送到肾小管，再通过重吸收全部回到体内，故其判断肾小管损伤的敏感性极高。近年临床研究[29-32]显示T2DM患者的uKIM-1、uNGAL、uRBP排泄水平均上升，并有学者[33]指出肾小管损伤标志物的异常对于肾功能损伤可能较蛋白尿出现更早，其特异性和敏感性更佳，甚至有研究[34]发现DKD患者尿液中肾小管损伤标志物水平可能较急性肾损害的患者更高。
本研究发现T2DM合并肾损伤患者Macro组uVDBP/Cr、uKIM-1/Cr、uNGAL/Cr及uRBP/Cr均较Nomo组及Micro组显著上升，uKIM-1/Cr、uNGAL/Cr、uRBP/CR与uVDBP/Cr均存在显著正相关，KIM-1、NGAL主要是由受损的肾小管上皮细胞生成，因而可能代表着肾小管细胞损伤而分泌增加[35]，而uVDBP/Cr及uRBP/Cr水平上升是因为肾小管重吸收功能障碍。其中uVDBP/Cr与uRBP/Cr的相关系数最大，可能是因为RBP与VDBP均为megalin的配体，在megalin及其共受体cubilin介导下重吸收[36]，并且在敲除megalin基因的小鼠模型中也发现小鼠的uVDBP及uRBP均增加[37]。
在区分UACR>30 mg/g及eGFR<60 mL/(min·1.73 m-2)的T2DM患者能力上，uVDBP/Cr均表现出较好的判别效能，提示uVDBP/Cr对T2DM患者的早期肾功能损害具有预测意义，并与疾病的进展有关。但本研究属于单中心研究，样本量偏少，须在后续研究中扩大样本量并进行基础研究证实该结论。
基金资助
国家自然科学基金(81870498)；湖南省自然科学基金(2021JJ31016)。
This work was supported by the National Natural Science Foundation (81870498) and the Natural Science Foundation of Hunan Province (2021JJ31016), China.
利益冲突声明
作者声称无任何利益冲突。
作者贡献
黄雨茜 研究构思，样本和数据收集，论文撰写；陈斯洁、戴晴 数据收集、样本处理。张浩、刘妍 论文构想和修改。所有作者阅读并同意最终的文本。
原文网址
http://xbyxb.csu.edu.cn/xbwk/fileup/PDF/20230140.pdf
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