Suzuki D, Yagame M, Kim Y, Sakai H, Mauer M
Division of Nephrology and Metabolism, Department of Internal Medicine, School of Medicine, Tokai University, Isehara, Kanagawa, Japan.
Nephron. 2002;92(3):564-72. doi: 10.1159/000064110.
BACKGROUND/AIM: Progressive expansion of mesangial matrix and glomerular basement membrane thickening represent alterations in the balance between synthesis and degradation of glomerular extracellular matrix (ECM) protein and are hallmarks of diabetic nephropathy. In order to elucidate the basis for this imbalance between the synthesis and the degradation of ECM in renal tissues from patients of type 1 diabetes mellitus (type 1D) with diabetic nephropathy (DN), we examined the expression of alpha1 chain of type IV collagen (IV-C), matrix metalloproteinase-2 and -3 (MMP-2, MMP-3), tissue inhibitor of metalloproteinase-1 (TIMP-1) and beta-actin mRNA using a high-resolution in situ hybridization with digoxigenin-labeled oligonucleotide.
Patients were divided into two groups based on both of degree of mesangial expansion using electron microscopic point counting morphometric methods and duration of type 1D: 7 'fast-track' patients were selected for their very rapid development of DN structural changes and 8 'slow-track' patients for their very slow development of DN structural changes. Seven normal human kidney (NHK) tissues were used as controls.
Positive cells for each mRNA were observed in glomerular resident cells, including glomerular mesangial, epithelial and endothelial cells and cells of Bowman's capsule. The percentage of glomerular cells positive for IV-C, MMP-2 and MMP-3 mRNA was significantly greater in the 'slow-track' vs. 'fast-track' patients. No significant differences in percentage positive cells was seen for beta-actin mRNA. Furthermore, to elucidate the total number of positive cells per glomerulus for each mRNA, we estimated total cell number of glomerulus using morphometric techniques on light microscopy tissues. The total cell number per glomerulus was significantly greater in 'fast-track' than that in 'slow-track' patients and NHK. The total number of positive cells per glomerulus for MMP-2 in NHK was significantly greater than that in 'slow-track' and 'fast-track' patients.
Thus, IV-C, MMP-2, MMP-3 and TIMP-1 mRNA are expressed in resident glomerular cells in renal tissues from NHK and type 1D. Glomerular alterations in these in situ mRNA expressions sufficient to explain ECM accumulation and DN risk were not uncovered. These largely negative results could be due to methodologic quantitative imprecision or could indicate that post-translational differences account for ECM imbalance in DN. However, these studies make it clear that unraveling the nature of the ECM production/removal imbalance in DN will require careful consideration of alterations in glomerular cell number.
背景/目的：系膜基质的进行性扩张和肾小球基底膜增厚代表肾小球细胞外基质（ECM）蛋白合成与降解平衡的改变，是糖尿病肾病的特征。为了阐明1型糖尿病（1D）合并糖尿病肾病（DN）患者肾组织中ECM合成与降解失衡的基础，我们使用地高辛标记的寡核苷酸进行高分辨率原位杂交，检测了IV型胶原α1链（IV-C）、基质金属蛋白酶-2和-3（MMP-2、MMP-3）、金属蛋白酶组织抑制剂-1（TIMP-1）和β-肌动蛋白mRNA的表达。
根据系膜扩张程度（采用电子显微镜点计数形态计量学方法）和1D病程将患者分为两组：7例“快速进展型”患者因DN结构变化发展非常迅速而入选，8例“缓慢进展型”患者因DN结构变化发展非常缓慢而入选。7例正常人类肾脏（NHK）组织用作对照。
在肾小球固有细胞中观察到每种mRNA的阳性细胞，包括肾小球系膜细胞、上皮细胞、内皮细胞和鲍曼囊细胞。“缓慢进展型”患者中IV-C、MMP-2和MMP-3 mRNA阳性的肾小球细胞百分比显著高于“快速进展型”患者。β-肌动蛋白mRNA阳性细胞百分比无显著差异。此外，为了阐明每个肾小球中每种mRNA阳性细胞的总数，我们在光学显微镜组织上使用形态计量技术估计肾小球的总细胞数。“快速进展型”患者每个肾小球的总细胞数显著多于“缓慢进展型”患者和NHK。NHK中MMP-2每个肾小球的阳性细胞总数显著多于“缓慢进展型”和“快速进展型”患者。
因此，IV-C、MMP-2、MMP-3和TIMP-1 mRNA在NHK和1D患者肾组织的肾小球固有细胞中表达。未发现这些原位mRNA表达的肾小球改变足以解释ECM积累和DN风险。这些大多为阴性的结果可能是由于方法学定量不精确，或者可能表明翻译后差异是DN中ECM失衡的原因。然而，这些研究清楚地表明，要弄清楚DN中ECM产生/清除失衡的本质，需要仔细考虑肾小球细胞数量的改变。
