We examined glomerular basement membrane anionic site distribution identified by cationic gold in seven patients with insulin-dependent and four patients with non-insulin-dependent diabetes mellitus, presenting a spectrum of clinical and glomerular changes. Anionic sites were investigated by pretreatment of tissue with glycosaminoglycan-degrading enzymes prior to cationic gold staining. The distribution of chondroitin sulphate proteoglycans--a previously unrecognized glomerular basement membrane component--and type IV collagen was examined by immunoelectron microscopy to identify structural changes in the basement membrane. Findings were compared with those of non-diabetic patients showing minor proteinuria and morphologically normal glomerular basement membranes. Two patients, originally diagnosed as having diabetic nephropathy were also examined at 19 weeks and 5 years after renal transplantation. Characteristic redistribution of type IV collagen and chondroitin sulphate proteoglycans was noted in thickened glomerular basement membrane segments (> 400 nm) of diabetic patients and those with renal transplants. Extension of anionic sites deep into the glomerular basement membrane at pH 2.5, together with loss of interna sites at pH 5.8 is unique to diabetic nephropathy. Reduced charge density was apparent in some patients due to thickening of the glomerular basement membrane, although the number of anionic sites per unit length of membrane was actually increased. Thus, charge aberration in diabetic nephropathy is due to displacement rather than loss of anionic sites. Removal of more than 90% of these sites by heparitinase, confirms their association with heparan sulphate proteoglycans. Similar derangement of anionic sites in all patients with diabetic nephropathy irrespective of the degree of proteinuria, suggests that a heparan sulphate proteoglycan-related charge barrier plays a minor role in controlling permeability of the diabetic glomerular basement membrane.

We detected glomerula.r anionic sites in fixed, LR Goldembedded ultra-thin tissue sections using cationic colloidal gold. Manual and computer-assisted quantitation were compared, and the influence of pH and glycosaminoglycandegrading enzymes on site expression was examined. Both quantitation methods produced similar results. Alteration ofpH within a narrow range (pH 2.5-3.0) markedly affected the staining pattern. At pH 2.5, epithelial and endothelial glycocalyx and regular sites restricted to the laming rara externa were stained. At pH 3.0 and above, glycocalyx was unstained but intracellular and nudear staining was present; glomerular basement membrane (GBM) and mesangial matnix sites were abundant. After chondroitinase ABC or hyaluronidase digestion, GBM staining was eliminated at pH 2.0 and reduced at pH 7.0 (p

Investigations of glomerular anionic charge status in human renal biopsies have previously been restricted, by the techniques and markers used, to staining of sites in pre-embedded tissue. The introduction of a novel marker, cationic colloidal gold, which demonstrates fixed anionic sites in hydrophilic resin (LR Gold)-embedded, ultrathin tissue sections, has now enabled glomerular charge to be evaluated in routine biopsy material. The cationic gold marker detects components which express anionic charge under different pH conditions. The patterns of staining in tissue showing minor glomerular pathology and low proteinuria, together with enzyme-digestion studies indicate that anionic sites are normally associated with heparan sulphate proteoglycans, glycocalyx sialoproteins, hyaluronic acid, and other GBM components which have not yet been characterised. Several charge aberrations involving different pathological mechanisms have been identified using cationic gold. These aberrations may be categorised according to the pathological basis of the charge pattern defect, rather than glomerular disease classification, as a prelude to the precise identification of the anionic sites and their functional importance in relation to the glomerular charge selectivity barrier. The categories which have been defined are: (1) 'Normal', (2) interrupted, (3) neutralised, (4) structurally disorganised, and (5) depleted. As sites are further characterised sub-categorisation is likely. We anticipate that this approach will help to elucidate both the participation of charged components in disease pathogenesis and their role in relation to glomerular proteinuria.

Elevated alpha-actinin-4 expression is observed only in MGN, and only in areas of subepithelial deposits. Further investigation into the cause of this may reveal insights into the pathogenesis of acquired nephrosis.

We assessed the relationship of certain clinical variables (including bradykinin [BK] release and dialysis membrane) to initial mean arterial pressure (MAP) reduction in 47 patients requiring continuous renal replacement therapy (CRRT) in an intensive care unit. The pretreatment MAP was 84 +/- 14 mm Hg for the group as a whole. The initial MAP reduction was 11.5 (7-20) mm Hg, occurring 4 to 8 min after connection. MAP reduction was 9 (6-15) mm Hg with polyacryonitrile (PAN) membranes versus 14 (5-19) mm Hg with polysulfone (PS) (not significant). There were positive correlations between MAP reduction and BK concentration at 3 (BK3; r = 0.58, p < 0.01) and 6 (BK6; r = 0.67, p < 0.001) min with PAN but not with PS. A greater reduction in MAP was seen in patients who were not receiving inotropic support (Mann-Whitney test, p < 0.01). BK3 and BK6 values for the PAN and PS groups were not significantly different. However, BK concentrations greater than 1,000 pg/ml were only seen with PAN (6 patients, MAP reduction 27 [17-31] mm Hg). There were positive (albumin) and negative (age; acute physiology, age, and chronic health evaluation score; C-reactive protein [CRP]; calcium) correlations with BK3/BK6 in the PAN and PS groups, some of which (albumin, CRP) reached statistical significance. In summary, MAP reduction at the start of CRRT correlates with BK concentration. The similarity of response with PAN and PS suggests an importance for other clinical factors. In this study, hemodynamic instability was more likely in patients with evidence of a less severe inflammatory or septic illness.