Experimental animal models indicate that complement contributes to tissue damage during brain ischaemia and stroke, but limited data are available for a role of the complement in human stroke. We, therefore, evaluated whether acute ischaemia leads to complement activation in human brain. Indirect immunohistochemical staining was performed on paraffin‐embedded, formalin‐fixed human brain from 10 patients and 10 controls. Complement components C1q, C3c and C4d were detected in all ischaemic lesions, suggesting activation via the classical pathway. C9, C‐reactive protein and IgM were detected in necrotic zones. Marked CD59 and weak CD55 expression were found in normal brains, but these complement regulators were virtually absent in ischaemic lesions. Modest amounts of mannose‐binding lectin (MBL), MBL‐associated serine protease‐2 and factor B were found in both ischaemic lesions and controls. These data suggest that increased deposition of complement components combined with decreased expression of complement regulators is a possible mechanism of tissue damage during ischaemia in human brain.

Inflammation plays a vital role in the development of diabetic nephropathy, but the underlying regulatory mechanisms are only partially understood. Our previous studies demonstrated that, during acute inflammation, endothelial heparan sulfate (HS) contributes to the adhesion and transendothelial migration of leukocytes into perivascular tissues by direct interaction with l-selectin and the presentation of bound chemokines. In the current study, we aimed to assess the role of endothelial HS on chronic renal inflammation and fibrosis in a diabetic nephropathy mouse model. To reduce sulfation of HS specifically in the endothelium, we generated Ndst1f/fTie2Cre+ mice in which N-deacetylase/N-sulfotransferase-1 (Ndst1), the gene that initiates HS sulfation modifications in HS biosynthesis, was expressly ablated in endothelium. To induce diabetes, age-matched male Ndst1f/fTie2Cre− (wild type) and Ndst1f/fTie2Cre+ mice on a C57Bl/6J background were injected intraperitoneally with streptozotocin (STZ) (50 mg/kg) on five consecutive days (N = 10–11/group). Urine and plasma were collected. Four weeks after diabetes induction the animals were sacrificed and kidneys were analyzed by immunohistochemistry and qRT-PCR. Compared to healthy controls, diabetic Ndst1f/fTie2Cre− mice showed increased glomerular macrophage infiltration, mannose binding lectin complement deposition and glomerulosclerosis, whereas these pathological reactions were prevented significantly in the diabetic Ndst1f/fTie2Cre+ animals (all three p < 0.01). In addition, the expression of the podocyte damage marker desmin was significantly higher in the Ndst1f/fTie2Cre− group compared to the Ndst1f/fTie2Cre+ animals (p < 0.001), although both groups had comparable numbers of podocytes. In the cortical tubulo-interstitium, similar analyses show decreased interstitial macrophage accumulation in the diabetic Ndst1f/fTie2Cre+ animals compared to the diabetic Ndst1f/fTie2Cre− mice (p < 0.05). Diabetic Ndst1f/fTie2Cre+ animals also showed reduced interstitial fibrosis as evidenced by reduced density of αSMA-positive myofibroblasts (p < 0.01), diminished collagen III deposition (p < 0.001) and reduced mRNA expression of collagen I (p < 0.001) and fibronectin (p < 0.001). Our studies indicate a pivotal role of endothelial HS in the development of renal inflammation and fibrosis in diabetic nephropathy in mice. These results suggest that HS is a possible target for therapy in diabetic nephropathy.

In Type 1 diabetes mellitus all tissues are exposed to hyperglycaemia, but only a subset (30±40 %) of diabetic patients develop diabetic kidney disease [1,2]. According to the Steno hypothesis, albuminuria and associated complications result from a genetic polymorphism of enzymes involved in the metabolism of heparan sulphate proteoglycans (HSPG) [2].HSPG consists of a core protein with heparan sulphate (HS) glycosaminoglycan side chains. Sulphate Diabetologia (1998) Summary In diabetic nephropathy, expression of glycosaminoglycan side chains of heparan sulphate proteoglycan in the glomerular basement membrane is reduced proportionally to the degree of proteinuria. We performed a cross-sectional study to evaluate whether non-vascular basement membranes also show a decrease in heparan sulphate side chain staining in patients with diabetic nephropathy. We evaluated the skin basement membrane for extracellular matrix components in the following groups: control subjects (n = 16); patients with Type 1 diabetes and normoalbuminuria (n = 17), microalbuminuria (n = 7), and macroalbuminuria (n = 16); patients with Type 1 diabetes and diabetic nephropathy undergoing renal replacement therapy (n = 13); and non-diabetic patients undergoing renal replacement therapy (n = 21). The following antibodies were used for this immunohistochemical study: monoclonal antibodies against the heparan sulphate side chain (JM403) and core protein (JM72) of the glomerular heparan sulphate proteoglycan; polyclonal antibodies against the core protein (B31); polyclonal antibodies against collagen types I, III, and IV, fibronectin, and laminin; and monoclonal antibodies against the noncollagenous domain of a1(collagen IV) and a3(collagen IV), against transforming growth factor b(2G7), and against advanced glycosylation end products (4G9). Expression of heparan sulphate side chains was reduced in the skin basement membrane of patients with overt diabetic nephropathy, of those with Type 1 diabetes undergoing renal replacement therapy, and those with non-diabetic renal failure. Increased intensity of staining was found for collagen type I and advanced glycosylation end products in patients with diabetic nephropathy. Changes in the extracellular matrix of the skin basement membrane seem to be similar to those in the glomerular basement membrane. These findings support the suggestion that patients with diabetic nephropathy also have altered heparan sulphate and collagen staining in extrarenal basement membranes. However, patients with non-diabetic renal failure also had reduced expression of heparan sulphate in the skin basement membrane, suggesting that this finding is not specific for diabetic nephropathy. [Diabetologia (1998) Keywords Advanced glycosylation end products, albuminuria, basement membrane, collagen, Type 1 diabetes mellitus, diabetic nephropathy, epidermodermal junction basement membrane zone, fibroblasts, heparitin sulphate, skin.Received: 13 November 1997 and in revised form: 10 February 1998Corresponding author: Dr. J. W. van de...

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