Menè P, Caenazzo C, Pugliese F, Cinotti G A, D'Angelo A, Garbisa S, Gambaro G
Department of Clinical Sciences, Division of Nephrology, University La Sapienza, Rome, Italy.
Nephrol Dial Transplant. 2001 May;16(5):913-22. doi: 10.1093/ndt/16.5.913.
Monocytes bind to human mesangial cells (HMC) in a co-culture model of leukocyte/ glomerular cell interactions. Since monocytic infiltration has been demonstrated in the early stages of diabetic glomerulopathy, we examined whether co-culture with myelomonocytes of the U937 cell line in media mimicking the diabetic microenvironment modulated phenotype, growth, and extracellular matrix production patterns of HMC.
HMC monolayers grown for 5 days in 5.5 mmol/l (NG) or 30 mmol/l (HG) glucose media were examined 3, 24 and 48 h after addition of U937 cells by computer-assisted image analysis/fluorescence microscopy following fixation, staining for cell adhesion, and TUNEL/propidium iodide labelling for apoptosis. As matrix components may be relevant to both phenotype of cultured HMC and monocyte adhesion, reverse transcription-polymerase chain reaction, zymography, and ELISA were used to detect urokinase-plasminogen activator (uPa), collagen type IV (COL IV), transforming growth factor beta1 (TGF-beta1), matrix metalloproteinases (MMP), and relative inhibitors (tissue inhibitor of MMP (TIMP)) expression in co-cultures in NG/HG.
U937 adhesion at 1-3 h was increased in HG (from 54.9+/-6.6 to 87.1+/-5.8% U937/HMC). Control HMC proliferating in NG supplemented with 10% fetal bovine serum had an average cross-sectional area of 9993+/-505 micro(2) with 1.2+/-0.1 hillocks/high-power field, which increased to 13 651+/- 1114 micro(2) with 0.5+/-0.2 hillocks/high-power field in HG (P<0.05). TUNEL+HMC were nearly identical (4.9+/-1.7 vs 4.2+/-0.4% in HG, P=NS). Enhanced transcription and secretion of urokinase (uPA, +656%), COL IV (+137%), TGF-beta1 (+590%) were observed in co-cultures in HG. COL IV and TGF-beta1, but not uPA, were also increased in HMC alone, exposed to HG for 5 days. MMP-2/TIMP-2 ratio was decreased while MMP-1/TIMP-1 was increased in HG co-cultures. In both NG and HG, U937 adhesion reduced HMC number and hillocks at 24 h, with constant apoptosis. The effects of U937 were no longer detectable at 48 h, when apoptosis was 2.1+/-0.6 vs 4.0+/-0.4% in HG, and cell counts returned above basal, possibly due to a delayed proliferative response.
High glucose medium increases U937 cell adhesion to HMC. In turn, monocytes modulate number and spatial distribution of HMC, which are also markedly affected by ambient glucose levels. These interactions may be relevant to leukocyte infiltration, mesangial expansion, and glomerulosclerosis in diabetes.
在白细胞/肾小球细胞相互作用的共培养模型中，单核细胞可与人系膜细胞（HMC）结合。鉴于在糖尿病肾小球病早期已证实存在单核细胞浸润，我们研究了在模拟糖尿病微环境的培养基中，U937细胞系的骨髓单核细胞与HMC共培养是否会调节HMC的表型、生长及细胞外基质产生模式。
在5.5 mmol/l（正常血糖，NG）或30 mmol/l（高血糖，HG）葡萄糖培养基中培养5天的HMC单层细胞，在加入U937细胞后3、24和48小时，通过计算机辅助图像分析/荧光显微镜进行检测，检测前先进行固定、细胞黏附染色以及TUNEL/碘化丙啶凋亡标记。由于基质成分可能与培养的HMC表型及单核细胞黏附均相关，因此采用逆转录-聚合酶链反应、酶谱分析和酶联免疫吸附测定法检测NG/HG共培养体系中尿激酶型纤溶酶原激活剂（uPa）、IV型胶原（COL IV）、转化生长因子β1（TGF-β1）、基质金属蛋白酶（MMP）及其相关抑制剂（基质金属蛋白酶组织抑制剂（TIMP））的表达。
HG条件下，1 - 3小时时U937细胞的黏附增加（从54.9±6.6% U937/HMC增加至87.1±5.8%）。在添加10%胎牛血清的NG培养基中增殖的对照HMC，其平均横截面积为9993±505μm²，每高倍视野有1.2±0.1个小丘，而在HG条件下，平均横截面积增加至13651±1114μm²，每高倍视野有0.5±0.2个小丘（P<0.05）。TUNEL阳性的HMC数量几乎相同（HG条件下分别为4.9±1.7%和4.2±0.4%，P=无显著差异）。在HG共培养体系中，观察到尿激酶（uPA）、COL IV、TGF-β1的转录和分泌增强（分别增加656%、137%、590%）。单独暴露于HG 5天的HMC中，COL IV和TGF-β1也增加，但uPA未增加。HG共培养体系中MMP-2/TIMP-2比值降低，而MMP-1/TIMP-1比值增加。在NG和HG条件下，U937细胞黏附均会在24小时时减少HMC数量和小丘数量，且凋亡持续存在。48小时时，U937细胞的影响不再明显，此时HG条件下的凋亡率为2.1±0.6%，而之前为4.0±0.4%，细胞计数恢复至基础水平以上，这可能是由于增殖反应延迟所致。
高糖培养基可增加U937细胞与HMC的黏附。反过来，单核细胞可调节HMC的数量和空间分布，而这也受到周围葡萄糖水平的显著影响。这些相互作用可能与糖尿病中的白细胞浸润、系膜扩张和肾小球硬化有关。
