背景
肾脏微循环由微动脉、微静脉和毛细血管组成，负责代谢废物清除、血压调节和电解质平衡维持。由于其结构复杂且代谢需求高，肾脏微循环易在缺血或缺氧状态下发生损伤，进而导致功能障碍。糖尿病肾病（diabetic kidney disease, DKD）是2型糖尿病患者最常见的微血管并发症之一。血管活性介质异常释放、肾素-血管紧张素-醛固酮系统激活以及糖基化终末产物积累等病理因素共同介导肾脏微血管结构重构、内皮细胞功能障碍及微血流动力学失衡，进一步加速DKD病程进展。因此，早期识别微循环功能障碍对保护糖尿病患者肾功能并改善DKD预后具有重要临床意义。鉴于肾脏微循环网络的复杂性及现有检测技术的局限性，建立可视化评估肾脏微循环功能的技术体系已成为亟待解决的科学问题。本研究旨在建立一种肾脏微循环功能测评、分析及可视化方法体系，通过不同遗传背景、不同性别小鼠肾脏微血流动力学特征验证该评估方法的有效性，并应用于探究2型糖尿病小鼠肾脏微循环障碍的病理表型，评估利拉鲁肽和胰岛素等降糖药物改善肾脏微循环功能的效应及潜在作用机制，同时分析不同种族人群中影响DKD发生风险的肾脏微循环关键因素。

方法
1、应用激光多普勒血流灌注监测仪分别检测雌雄BALB/c、C57BL/6和KM小鼠肾脏微血流动力学参数；采用小波变换分析肾脏微血管血流灌注、血流速度及红细胞密度信号在一氧化氮依赖性和非依赖性内皮细胞源幅值、神经源性幅值、肌源性幅值、呼吸源性幅值及心源性幅值；通过Mantel和Pearson相关分析建立并比较不同遗传背景、不同性别小鼠血清肾功能指标、心率及血压与肾脏微循环功能的关联；采用免疫组织化学及HE染色技术分析不同品系、不同性别小鼠肾脏微血管内皮细胞功能蛋白CD31、雌激素受体α、β表达水平及肾脏微血管形态与密度。
2、应用微循环增强灌注血氧监测系统（enhanced perfusion and oxygen saturation system, EPOS）同步采集对照组、T2DM模型组、T2DM+胰岛素治疗组（1周或2周）、T2DM+利拉鲁肽治疗组（1周或2周）小鼠肾脏微循环氧代谢指标（总血红蛋白、氧合血红蛋白、还原血红蛋白、氧饱和度、红细胞组织分数）和微血流动力学参数（速度分级血流灌注量、总血流灌注量、常规血流灌注量），并进行三维可视化建模；比较各组小鼠血清肾功能指标差异，利用Pearson检验分析血清肾功能指标与肾脏微循环参数的相关性；分离、纯化、鉴定和培养小鼠原代肾脏微血管内皮细胞，分别给予正常糖、高糖、高糖+胰岛素（24 h或48 h）、高糖+利拉鲁肽（24h或48h）条件下培养，通过细胞划痕实验及血管形成实验比较各组细胞的迁移能力和血管生成能力；采用非靶向代谢组学筛选和比较各组小鼠肾脏代谢物谱差异，进一步通过氨基酸靶向代谢组学精确定量不同组别间氨基酸代谢水平变化，建立氨基酸代谢与肾脏微循环功能的关联网络。
3、分析“中国健康与养老追踪调查”1115名和“英国老龄化纵向研究”中111名糖尿病患者的临床资料。将血红蛋白水平按照各数据库中人群分布特征分为三等分，采用统一标准定义DKD，包括估计肾小球滤过率、尿蛋白定量评估和自我报告结果。在调整人口学特征、临床指标和生活方式等因素后，采用COX回归模型分析血红蛋白水平与DKD发生风险的关联。

结果
1、通过小波分析二维频谱图和三维时频图，解析了肾脏微血管血流灌注、血流速度及红细胞密度信号在肾脏微循环功能相关的6种生理性振荡频率特征及节律变化，实现了肾脏微循环功能的可视化评估。BALB/c、C57BL/6和KM品系的雌雄小鼠肾脏微血流动力学表现出明显异质性。在肾脏微循环血流灌注方面，BALB/c雌性小鼠的一氧化氮非依赖性内皮源性振幅高于雄性（P = 0.021）；在红细胞聚集度方面，KM雄性小鼠的一氧化氮非依赖性内皮源性振幅显著高于KM雌性小鼠（P = 0.029）。三种品系的雌雄小鼠微循环血流速度的六种生理性振幅均未见显著差异（P均 > 0.05）。此外，雌性KM小鼠的肾脏微血管阻力指数显著高于雄性（P = 0.0002），而BALB/c及C57BL/6小鼠未见显著差异。Pearson相关分析表明，血清葡萄糖水平与血流灌注的心源性（r = 0.410；P = 0.013）和呼吸源性振幅（r = 0.381; P = 0.022）呈显著正相关，Mantel检验表明BALB/c雌性小鼠的微循环幅值与血清肌酐（r = 0.603; P = 0.047）及尿酸（r = 0.817; P = 0.033）水平具有相关性。HE染色及免疫组化结果表明雌性小鼠肾脏微血管密度显著高于雄性，KM小鼠肾脏微血管ERβ表达水平高于C57BL/6小鼠。不同遗传背景、不同性别小鼠的大循环与微循环相关模式存在异质性。
2、对照组、T2DM模型组及不同治疗干预组小鼠肾脏微循环氧代谢指标存在显著差异。与T2DM组相比，胰岛素治疗1周组小鼠肾脏的红细胞组织分数、总血红蛋白和还原血红蛋白水平降低，氧饱和度升高（P = 0.002, P = 0.002, P = 0.0004和P = 0.008）。胰岛素治疗2周组、利拉鲁肽治疗1周及2周组小鼠肾脏微循环红细胞组织分数及总血红蛋白较T2DM组显著增加（P < 0.0001, P < 0.0001, P < 0.002）；此外，氧合血红蛋白水平亦较T2DM组显著增加（P < 0.0004, P < 0.0001, P < 0.0004）。胰岛素治疗2周组较1周组表现出更高的红细胞组织分数、总血红蛋白、氧合和还原血红蛋白水平（P均 < 0.0001），以及更低的氧饱和度（P = 0.009）。利拉鲁肽治疗1周和2周组间微循环氧代谢指标未见显著差异（P均 > 0.05）。此外，各组间肾脏微血流动力学参数未发现显著差异。
与对照组相比，T2DM小鼠的血清肌酐浓度显著升高（P = 0.029）。与未经治疗的T2DM组相比，胰岛素治疗（1周和2周）和利拉鲁肽治疗（1周）显著降低了肌酐水平（P = 0.0009、P = 0.001和P < 0.0001）；胰岛素和利拉鲁肽治疗组的尿素水平较T2DM组显著降低（治疗1周和2周P均 < 0.001）。胰岛素治疗2周后胱抑素C水平高于T2DM组（P = 0.027）。肾功能与微循环指标相关性分析表明，血糖水平与红细胞组织分数/总血红蛋白（P = 0.024, r = - 0.328）、氧合血红蛋白（P = 0.039，r = - 0.300）和还原血红蛋白（P = 0.022, r = - 0.331）等肾脏微循环功能指标呈负相关。尿素水平升高与氧饱和度下降（P = 0.024, r = - 0.325）、各速度分级血流灌注量减少（<1 mm/s: P = 0.036, r = - 0.303; 1-10 mm/s: P = 0.001, r = - 0.450; >10 mm/s: P = 0.0009, r = - 0.466）、总血流灌注量（P = 0.0005, r = - 0.482）及常规血流灌注量（P = 0.0002; r = - 0.509）降低显著相关。
成功分离、鉴定并纯化小鼠原代肾脏微血管内皮细胞（renal microvascular endothelial cells, RMECs）。划痕实验表明，划痕形成48小时后，对照组、胰岛素组和利拉鲁肽组RMECs迁移距离较高糖组显著增加（P均 < 0.0001）。血管形成实验结果显示，与对照组相比，高糖条件下RMECs的覆盖面积、总分支点数和总管长度显著降低（P = 0.006、P = 0.002 和 P = 0.016），而总网络数增加（P = 0.015），表明RMECs血管生成功能受损。与高糖组相比，利拉鲁肽（20 nM）处理48小时后，RMECs覆盖面积、总血管数、总分支点数和总管长度显著增加，总网络数减少（P = 0.002、P = 0.0002、P = 0.010、P = 0.004 和 P = 0.009）。与处理24小时相比，利拉鲁肽处理48小时组RMECs覆盖面积和总分支点数进一步增加（P = 0.039、P = 0.029）。与胰岛素处理48小时组相比，利拉鲁肽处理48小时组表现出更多的总分支点数和更少的总网络数（P = 0.012、P = 0.005）。胰岛素处理组与高糖组相比未观察到显著差异。
非靶向代谢组学分析表明，T2DM组小鼠肾脏存在明显的代谢紊乱，KEGG通路富集分析显示差异代谢物主要富集于“氨基酸代谢”通路。靶向氨基酸代谢分析结果表明，与对照组相比，T2DM组的谷氨酰胺、苯丙氨酸、蛋氨酸、色氨酸及鸟氨酸含量显著降低，而利拉鲁肽治疗后谷氨酰胺、苯丙氨酸、蛋氨酸、色氨酸含量显著上升。Spearman相关性分析表明，苯丙氨酸、鸟氨酸、蛋氨酸及谷氨酰胺含量与红细胞组织分数/总血红蛋白、氧合血红蛋白水平呈显著正相关。
3、在中国和英国两个队列中，与低血红蛋白水平组相比，中等血红蛋白水平组（CHARLS：HR = 0.49，95 % CI：0.32-0.76，P = 0.002；ELSA：HR = 0.20，95 % CI：0.05-0.75，P = 0.017）和高血红蛋白水平组（CHARLS：HR = 0.43，95 % CI：0.27-0.69，P < 0.001；ELSA：HR = 0.14，95 % CI：0.03-0.76，P = 0.023）的DKD发生风险均显著降低。

结论 
1、本研究基于激光多普勒技术和小波变换分析建立了系统性肾脏微循环功能评估框架，为肾脏微循环功能的定量测量和动态解析提供了新方法。BALB/c、C57BL/6J及KM品系雌雄小鼠的肾脏微循环功能具有遗传背景异质性和性别二态性，且肾脏微循环功能与肾功能指标间存在显著相关性。
2、T2DM小鼠表现出明显的肾脏微循环功能障碍、代谢紊乱和肾脏微血管内皮细胞功能下降。与胰岛素相比，利拉鲁肽不仅能有效控制血糖，还能通过改善肾脏微血流动力学、优化微循环氧代谢、修复内皮细胞功能和调节关键代谢通路等多重机制发挥综合性肾脏保护作用。
3、在中国和英国两个不同人群队列中，血红蛋白水平与DKD发生风险呈负相关关系，中等和高水平血红蛋白是DKD的独立保护因素。血红蛋白可成为DKD早期识别、风险分层的候选指标。

Background
Renal microcirculation, consisting of arterioles, venules, and capillaries, is responsible for metabolic waste elimination, blood pressure regulation, and electrolyte balance maintenance. Due to its complex structure and high metabolic demands, renal microcirculation is vulnerable to injury under ischemic or hypoxic conditions, leading to functional impairment. Diabetic kidney disease (DKD) is one of the most common microvascular complications in type 2 diabetes patients. Pathological factors including abnormal release of vasoactive mediators, activation of the renin-angiotensin-aldosterone system, and accumulation of advanced glycation end products collectively mediate renal microvascular structural remodeling, endothelial dysfunction, and microcirculatory hemodynamic imbalance, further accelerating DKD progression. Therefore, early identification of microcirculatory dysfunction has significant clinical implications for protecting renal function and improving DKD prognosis in diabetic patients. Given the complexity of renal microcirculatory networks and limitations of existing detection technologies, establishing a technical system for visualizing and evaluating renal microcirculatory function has become an urgent scientific challenge. This study aims to establish a comprehensive method for evaluating, analyzing, and visualizing renal microcirculatory function, validate its effectiveness through microcirculatory hemodynamic characteristics in mice of different genetic backgrounds and sexes, investigate pathological phenotypes of renal microcirculatory dysfunction in type 2 diabetic mice, assess the effects and potential mechanisms of hypoglycemic drugs such as liraglutide and insulin in improving renal microcirculatory function, and analyze key microcirculatory factors affecting DKD risk in different ethnic populations.

Methods
1. The laser Doppler perfusion monitor was applied to detect renal microcirculatory hemodynamic parameters in female and male BALB/c, C57BL/6, and KM mice, wavelet transform analysis was used to analyze signal amplitudes of renal microvascular perfusion, blood flow velocity, and erythrocyte concentration in nitric oxide-dependent and non-dependent endothelial-derived, neurogenic, myogenic, respiratory, and cardiac origins, Mantel and Pearson correlation analyses were employed to establish and compare associations between serum renal function indicators, heart rate, blood pressure, and renal microcirculatory function across mice of different genetic backgrounds and sexes, immunohistochemistry and HE staining techniques were used to analyze the expression levels of endothelial functional proteins CD31, estrogen receptors α and β, and renal microvascular morphology and density in mice of different strains and sexes.
2. The enhanced perfusion and oxygen saturation system (EPOS) was utilized to simultaneously collect renal microcirculatory oxygen metabolism indicators (total hemoglobin, oxygenated hemoglobin, deoxygenated hemoglobin, oxygen saturation, erythrocyte tissue fraction) and microcirculatory hemodynamic parameters (velocity-graded blood perfusion, total blood perfusion, conventional blood perfusion) in control, T2DM model, T2DM+insulin treatment (1 or 2 weeks), and T2DM+liraglutide treatment (1 or 2 weeks) groups, followed by three-dimensional visualization modeling, serum renal function indicators were compared across groups, and Pearson tests were used to analyze correlations between serum renal function indicators and renal microcirculatory parameters, mouse primary renal microvascular endothelial cells were isolated, purified, identified, and cultured under normal glucose, high glucose, high glucose+insulin (24h or 48h), or high glucose+liraglutide (24h or 48h) conditions, with cell migration ability and angiogenic capacity compared through scratch assays and tube formation experiments, untargeted metabolomics was employed to screen and compare metabolic profile differences among groups, and targeted amino acid metabolomics was further used to accurately quantify amino acid metabolism level changes between different groups, establishing association networks between amino acid metabolism and renal microcirculatory function.
3. Clinical data from 1,115 diabetic patients in the China Health and Retirement Longitudinal Study and 111 diabetic patients in the English Longitudinal Study of Ageing were analyzed. Hemoglobin levels were divided into tertiles according to population distribution characteristics in each database, and DKD was defined using unified standards, including estimated glomerular filtration rate, quantitative urinary protein assessment, and self-reported results. COX regression models were employed to analyze the association between hemoglobin levels and DKD risk after adjusting for demographic characteristics, clinical indicators, and lifestyle factors.

Results
1. Through wavelet analysis of two-dimensional frequency spectra and three-dimensional time-frequency plots, we decoded the characteristics and rhythmic changes of renal microvascular perfusion, blood flow velocity, and erythrocyte concentration signals at six physiological oscillatory frequencies related to renal microcirculatory function, achieving visualization of renal microcirculatory function assessment. Female and male BALB/c, C57BL/6, and KM strain mice exhibited marked heterogeneity in renal microhemodynamics. Regarding renal microcirculatory perfusion, female BALB/c mice showed higher nitric oxide-independent endothelial-derived amplitude than males (P = 0.021). In terms of erythrocyte concentration, male KM mice exhibited significantly higher nitric oxide-independent endothelial-derived amplitude than female KM mice (P = 0.029). No significant differences were observed in the six physiological amplitudes of microcirculatory blood flow velocity among female and male mice of the three strains (all P > 0.05). Additionally, female KM mice demonstrated significantly higher renal microvascular resistance index than males (P = 0.0002), while no significant differences were observed in BALB/c and C57BL/6 mice. Pearson correlation analysis indicated that serum glucose levels were significantly positively correlated with cardiac-derived (r = 0.410; P = 0.013) and respiratory-derived amplitudes (r = 0.381; P = 0.022) of blood perfusion. Mantel tests revealed that microcirculatory amplitudes in female BALB/c mice were correlated with serum creatinine (r = 0.603; P = 0.047) and uric acid (r = 0.817; P = 0.033) levels. HE staining and immunohistochemistry results demonstrated that female mice had significantly higher renal microvascular density than males, and KM mice exhibited higher ERβ expression in renal microvessels than C57BL/6 mice. Heterogeneity existed in the correlation patterns between macro-circulation and micro-circulation among mice with different genetic backgrounds and sexes.
2. Significant differences in renal microcirculatory oxygen metabolism indicators were observed among control, T2DM model, and different treatment intervention groups. Compared with the T2DM group, mice treated with insulin for 1 week showed reduced erythrocyte tissue fraction, total hemoglobin, and deoxygenated hemoglobin levels, with increased oxygen saturation (P = 0.002, P = 0.002, P = 0.0004, P = 0.008). Mice treated with insulin for 2 weeks or liraglutide for 1 or 2 weeks exhibited significantly increased renal microcirculatory erythrocyte tissue fraction and total hemoglobin compared to the T2DM group (P < 0.0001, P < 0.0001, P < 0.002). Furthermore, oxygenated hemoglobin levels were also significantly increased compared to the T2DM group (P < 0.0004, P < 0.0001, P < 0.0004). The 2-week insulin treatment group demonstrated higher erythrocyte tissue fraction, total hemoglobin, and oxygenated and deoxygenated hemoglobin levels (all P < 0.0001), as well as lower oxygen saturation (P = 0.009) compared to the 1-week group. No significant differences in microcirculatory oxygen metabolism indicators were observed between the 1-week and 2-week liraglutide treatment groups (all P > 0.05). Additionally, no significant differences in renal microhemodynamic parameters were found among groups.
Compared with the control group, T2DM mice showed significantly elevated serum creatinine concentrations (P = 0.029). Compared with the untreated T2DM group, insulin treatment (1 and 2 weeks) and liraglutide treatment (1 week) significantly reduced creatinine levels (P = 0.0009, P = 0.001, and P < 0.0001); both insulin and liraglutide treatment groups exhibited significantly lower urea levels than the T2DM group (all P < 0.001). Cystatin C levels were higher in the 2-week insulin treatment group compared to the T2DM group (P = 0.027). Correlation analysis between renal function and microcirculatory indicators revealed that blood glucose levels were negatively correlated with renal microcirculatory function indicators such as erythrocyte tissue fraction/total hemoglobin (P = 0.024, r = -0.328), oxygenated hemoglobin (P = 0.039, r = -0.300), and deoxygenated hemoglobin (P = 0.022, r = -0.331). Elevated urea levels were significantly associated with decreased oxygen saturation (P = 0.024, r = -0.325), reduced velocity-resolved blood perfusion (<1 mm/s: P = 0.036, r = -0.303; 1-10 mm/s: P = 0.001, r = -0.450; >10 mm/s: P = 0.0009, r = -0.466), decreased total blood perfusion (P = 0.0005, r = -0.482), and reduced conventional blood perfusion (P = 0.0002; r = -0.509). 
Renal microvascular endothelial cells (RMECs) were isolated, identified, and purified. Scratch assays revealed that 48 hours after scratch formation, RMECs in the control, insulin, and liraglutide groups migrated significantly further than those in the high glucose group (all P < 0.0001). Tube formation assay results showed that compared with the control group, RMECs under high glucose conditions exhibited significantly reduced coverage area, total junction number, and total tube length (P = 0.006, P = 0.002, P = 0.016), with increased total in the total nets (P = 0.015), indicating impaired angiogenic function. Compared with the high glucose group, RMECs treated with liraglutide (20 nM) for 48 hours showed significantly increased coverage area, total tube number, total junction number, and total tube length, with decreased total network number (P = 0.002, P = 0.0002, P = 0.010, P = 0.004, P = 0.009). Compared with 24-hour treatment, 48-hour liraglutide treatment further increased RMECs covered area and total branching points (P = 0.039, P = 0.029). Compared to the 48-hour insulin-treated group, the liraglutide-treated group showed an increased total branching points and fewer total net (P = 0.012, P = 0.005). No significant differences were observed between the insulin treatment group and the high glucose group.
Untargeted metabolomics analysis revealed significant metabolic disturbances in the kidneys of T2DM group mice, with KEGG pathway enrichment analysis showing that differential metabolites were primarily enriched in the “amino acid metabolism” pathway. Targeted amino acid metabolic analysis results indicated that compared with the control group, the T2DM group had significantly decreased glutamine, phenylalanine, methionine, tryptophan, and ornithine contents, while liraglutide treatment significantly increased glutamine, phenylalanine, methionine, and tryptophan contents. Spearman correlation analysis demonstrated that phenylalanine, ornithine, methionine, and glutamine contents were significantly positively correlated with erythrocyte tissue fraction/total hemoglobin and oxygenated hemoglobin levels. 
3. In both Chinese and British cohorts, compared with the low hemoglobin level group, the medium hemoglobin level group (CHARLS: HR = 0.49, 95% CI: 0.32 - 0.76, P = 0.002; ELSA: HR = 0.20, 95% CI: 0.05 - 0.75, P = 0.017) and high hemoglobin level group (CHARLS: HR = 0.43, 95% CI: 0.27 - 0.69, P < 0.001; ELSA: HR = 0.14, 95% CI: 0.03 - 0.76, P = 0.023) had significantly lower risk of DKD occurrence.

Conclusion
1. This study established a systematic framework for renal microcirculatory function assessment based on laser Doppler technology and wavelet transform analysis, providing a novel method for quantitative measurement and dynamic analysis of renal microcirculatory function. Renal microcirculatory function in female and male BALB/c, C57BL/6J, and KM strain mice exhibited genetic background heterogeneity and sex dimorphism, with significant correlations between renal microcirculatory function and renal function indicators.
2. T2DM mice demonstrated significant renal microcirculatory dysfunction, metabolic disturbances, and impaired renal microvascular endothelial cell function. Compared with insulin, liraglutide not only effectively controlled blood glucose but also exerted renoprotective effects through multiple mechanisms, including improving renal microhemodynamics, optimizing microcirculatory oxygen metabolism, repairing endothelial cell function, and regulating key metabolic pathways.
3. In both Chinese and British population cohorts, hemoglobin levels were negatively correlated with DKD risk, with medium and high hemoglobin levels serving as independent protective factors against DKD. Hemoglobin could serve as a candidate indicator for early identification and risk stratification of DKD.