Diabetic nephropathy is characterized by albuminuria which proceeds to overt proteinuria. The highly negatively stained HS side chain of heparan sulphate proteoglycan (HSPG) is a major determinant of the charge-dependent permeability of the GBM. We set out to study the presence of HS and HSPG in the GBM of patients with diabetic nephropathy using newly developed monoclonal antibodies, and to compare HSPG expression to the expression of other previously investigated glomerular extracellular matrix compounds. Immunohistochemically, glomerular extracellular matrix components were analysed in 14 renal biopsies of patients with diabetic nephropathy and compared with those of normal control subjects. Monoclonal antibodies used were: JM403 against the HS side chain of GBM HSPG and JM72 against the HSPG-core protein. Also, a polyclonal antiserum (B31) against human GBM-HSPG-core protein was used. Additionally, antibodies were used against collagen types I, III, IV and against alpha 1 (IV)NC, alpha 3(IV)NC and fibronectin. Staining was scored for intensity and for staining pattern by four independent observers who had no previous knowledge of the sample origin. No glomerular staining was seen for collagen type I. Collagen type III was present in some diabetic nodules. Anti-collagen type IV showed a decreased GBM staining in patients with diabetic nephropathy (p = 0.04). With anti-alpha 1 (IV)NC no changes in GBM staining intensity were observed; with anti-alpha 3 (IV)NC brilliant GBM staining was seen in both groups. Increased mesangial staining (p = 0.003) was seen with anti-collagen type IV in biopsies with nodular lesions.(ABSTRACT TRUNCATED AT 250 WORDS)

Objective-In vitro studies implicate that the low-density lipoprotein receptor (LDLR)-related protein (LRP) in macrophages has a pro-atherogenic potential. In the present study, we investigated the in vivo role of macrophage specific LRP in atherogenesis independent of its role in the uptake of lipoproteins. Methods and Results-We generated macrophage-specific LRP-deficient mice on an apoE/LDLR double-deficient background. Macrophage LRP deletion did not affect plasma cholesterol and triglyceride levels, lipoprotein distribution, and blood monocyte counts. Nevertheless, macrophage LRP deficiency resulted in a 1.8-fold increase in total atherosclerotic lesion area in the aortic root of 18-week-old mice. Moreover, LRP deficiency also resulted in a relatively higher number of advanced lesions. Whereas macrophage and smooth muscle cell content did not differ between LRP-deficient mice and control littermates, a 1.7-fold increase in collagen content and 2.3-fold decrease in relative number of CD3ϩ T cells were observed in lesions from macrophage specific LRP-deficient mice. Conclusions-Our data demonstrate that independent of its role in lipoprotein uptake, absence of LRP in macrophages resulted in more advanced atherosclerosis and in lesions that contained more collagen and less CD3ϩ T cells. In contrast to previous in vitro studies, we conclude that macrophage LRP has an atheroprotective potential and may modulate the extracellular matrix in the atherosclerotic lesions. Key Words: atherosclerosis Ⅲ collagen Ⅲ genetically altered mice Ⅲ LRP Ⅲ macrophage C ardiovascular diseases are the leading cause of morbidity and mortality in the Western world. The primary cause of cardiovascular diseases is atherosclerosis, which is characterized by lipid accumulation and inflammation in the vascular wall. 1,2 Macrophages play a central role in the pathogenesis of atherosclerosis by internalizing modified low-density lipoprotein (LDL), production of cytokines and growth factors, and thus stimulate migration and proliferation of smooth muscle cells (SMCs), and plaque development and progression. 1 The LDL receptor (LDLR)-related protein (LRP) is a large-cell-surface multi-ligand endocytic clearance and signaling receptor of the LDLR gene family. [3][4][5][6][7][8] LRP is known to recognize Ͼ50 structurally and functionally different ligands. 9,10 It is expressed in a variety of cell types including hepatocytes, SMCs, and macrophages. 11 The hepatic LRP was originally identified as an endocytic receptor for apolipoprotein E (apoE)-rich lipoproteins. 4 Recently, we showed that hepatic LRP deficiency in mice increased atherosclerosis independent of plasma lipoproteins. 12 Similarly, SMCspecific LRP-deficient mice display impaired vessel wall integrity and have increased susceptibility to cholesterol-diet induced atherosclerosis. 7 These data show that LRP protects against the development of atherosclerosis at the level of the liver and the SMCs, independent of its role in the removal of plasma lipoproteins.In contrast, several lines...

Changes in heparan sulfate metabolism may be important in the pathogenesis of diabetic nephropathy. Recent studies performed on renal biopsies from patients with diabetic nephropathy revealed a decrease in heparan sulfate glycosaminoglycan staining in the glomerular basement membrane without changes in staining for heparan sulfate proteoglycan-core protein. To understand this phenomenon at the cellular level, we investigated the effect of high glucose conditions on the synthesis of heparan sulfate proteoglycan by glomerular cells in vitro. Human adult mesangial and glomerular visceral epithelial cells were cultured under normal (5 mM) and high glucose (25 mM) conditions. Immunofluorescence performed on cells cultured in 25 mM glucose confirmed and extended the in vivo histological observations. Using metabolic labeling we observed an altered proteoglycan production under high glucose conditions, with predominantly a decrease in heparan sulfate compared to dermatan sulfate or chondroitin sulfate proteoglycan. N-sulfation analysis of heparan sulfate proteoglycan produced under high glucose conditions revealed less di- and tetrasaccharides compared to larger oligosaccharides, indicating an altered sulfation pattern. Furthermore, with quantification of glomerular basement membrane heparan sulfate by ELISA, a significant decrease was observed when mesangial and visceral epithelial cells were cultured in high glucose conditions. We conclude that high glucose concentration induces a significant alteration of heparan sulfate production by mesangial cells and visceral epithelial cells. Changes in sulfation and changes in absolute quantities are both observed and may explain the earlier in vivo observations. These changes may be of importance for the altered integrity of the glomerular charge-dependent filtration barrier and growth-factor matrix interactions in diabetic nephropathy.

Accumulation of matrix proteins is a prominent feature of diabetic nephropathy. Glomerular visceral epithelial cells (GVECs) are important contributors to extracellular matrix (ECM) production in the glomerulus. Factors involved with increased accumulation of ECM proteins are high glucose, angiotensin II (ANG II), and transforming growth factor (TGF)-beta. Therefore, we investigated the effects of high glucose and ANG II on fibronectin and TGF-beta production by human GVECs in vitro. We found that ANG II had no effect on the production of fibronectin and TGF-beta by GVECs. Using reverse transcriptase-polymerase chain reaction analysis, no ANG II receptor could be detected on these cells. However, high glucose induced a twofold increase in fibronectin (P < 0.01) and a three- to sixfold increase in TGF-beta (P < 0.001) production. Similar results were obtained by analyzing the mRNA levels of fibronectin (increased 2.7-fold) and TGF-beta (increased 3.5-fold). Addition of increasing concentrations of rTGF-beta to control cells resulted in increased fibronectin production. Neutralizing antibodies against TGF-beta significantly reversed the increase in fibronectin protein and mRNA caused by high glucose back to control levels. We conclude that high glucose concentrations stimulate the synthesis of fibronectin and that this effect is mediated by induction of TGF-beta. These results suggest that in diabetic nephropathy, high glucose levels play a role in changing the matrix composition of the glomerular basement membrane through induction of TGF-beta. Our results indicate that a contribution to this process by an effect of ANG II on GVECs seems unlikely.

Decreased expression of heparan sulphate has been shown in the glomerular basement membrane of patients with over diabetic nephropathy. Low- molecular-weight heparin (LMWH) is a highly sulphated glycosaminoglycan with strong structural and functional similarities to heparan sulphate. In a first study, we set out to assess if LMWH could decrease the urinary albumin excretion rate (AER) in diabetic patients with over nephropathy. Six patients entered a randomized, double-blind, placebo-controlled crossover study with treatment episodes of 1 month, separated by a 1-month wash-out. Patients self-administered prefilled syringes with either placebo or LMWH (enoxaparin 40 mg/0.4 ml) at bedtime. Baseline AER levels before either treatment period were similar. In contrast to placebo, AER significantly decreased from 447 (181-1102) to 295 (100-873) micrograms/min after 1 month treatment with LMWH (P < 0.05). Compared to placebo, the effect of LMWH did not reach statistical significance in these six patients after 1 month treatment (P = 0.16). Haemodynamic variables including glomerular filtration rate and filtration fraction did not change during enoxaparin treatment. We observed a favourable effect on AER during LMWH treatment in diabetic patients with over nephropathy. These data suggest that long-term treatment trials in a larger group of patients may potentially demonstrate a new therapeutic option for patients with over diabetic nephropathy.