Renal in situ Hybridization Studies of Extracellular Matrix Related Molecules in Type 1 Diabetes mellitus

Abstract

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 α1 chain of type IV collagen (IV-C), matrix metalloproteinase-2 and -3 (MMP-2, MMP-3), tissue inhibitor of metalloproteinase-1 (TIMP-1) and β-actin mRNA using a high-resolution in situ hybridization with digoxigenin-labeled oligonucleotide. Methods: 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. Results: 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 β-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. Conclusion: 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.

Therapeutic Potential of Angiostatin in Diabetic Nephropathy

“…This conclusion is further supported by the observation that both the expression and the activity of MMP-2 are suppressed in diabetic kidney, as MMP-2 has been shown to release angiostatin from plasminogen (38). Although endothelial cells in the retina are recognized as a major source of MMP, previous studies showed that MMP are expressed mainly in mesangial cells and podocytes in the glomeruli (39,40). In the three types of glomerular cells, MMP-2 mRNA levels in the podocytes and endothelial cells were only 33 and 18%, respectively, of that in the mesangial cells (40).…”

“…As endothelial cells in the glomeruli express only very low levels of MMP, we first compared the MMP-2 activity in the cultured HMC and the podocyte cell line (39). The results showed that the MMP-2 activity in the conditioned medium from HMC was 10-fold higher than that from podocytes after a 48-h culture (P Ͻ 0.01; Figure 2D), consistent with the previous study showing the high expression of MMP-2 in mesangial cells but not in podocyte or endothelial cells (40).…”

Role of matrix metalloproteinases in renal pathophysiologies

“…In mouse kidney, TIMP-1 is expressed at low levels, TIMPs-2 and -3 at high levels, and TIMP-4 is not expressed (28). TIMP-1 is expressed in the glomerulus in rats (84) and humans (79,80). In addition, TIMP-3 is detected in the rat kidney (89), and human glomeruli express TIMP-1 and -2 mRNA (15).…”

Serglycin Is a Major Proteoglycan in Polarized Human Endothelial Cells and Is Implicated in the Secretion of the Chemokine GROα/CXCL1

“…Xyloside and PGs in HUVEC-To further address the role of serglycin in polarized HUVEC, we used ␤-D-xylosides, agents that are known to inhibit the assembly of intact PGs (22)(23)(24). HUVEC were cultured with or without 1 mM xyloside and were biosynthetically labeled with [ 35 S]sulfate.…”