The bradykinin response and early hypotension at the introduction of continuous renal replacement therapy in the intensive care unit.

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.

Expression of an antigen associated with basal bodies of human ciliated epithelial cells.

The process and regulation of ciliogenesis in human epithelia is little understood and many components of the cilium and associated structures have not been characterised. We have identified a monoclonal antibody, LhS28, which recognises a 44,000-45,000 M(r) protein specifically associated with human ciliated epithelial cells. Immunoperoxidase labelling of formalin-fixed paraffin wax-embedded human tissues showed that LhS28 was expressed in the sub-apical zone of ciliated epithelial cells of the Fallopian tube and upper respiratory tract, but not ciliated ependyma, non-ciliated epithelia or testis containing developing spermatozoa. Immunoelectron microscopy demonstrated that the antigen recognised by LhS28 was associated with the basal body structure of the cilium and specifically with the 9 + 0 microtubule arrays. LhS28 should be a useful tool in the identification of ciliated cells in pathological specimens and for investigating mechanisms of ciliogenesis.

Alterations of glomerular basement membrane charge and structure in diabetic nephropathy.

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.

Clinical features and outcome of patients with thin and ultrathin glomerular membranes.

There is considerable disagreement regarding the natural history of renal disease associated with thin glomerular basement membranes (TGBM). We followed 43 patients (19 male), mean age 41.6 years (range 19-73) for a mean of 88 months (48-140). TGBM was recognized in adults when glomerular basement membrane thickness, measured from multiple sites in electronmicrographs of renal biopsy tissue as the harmonic mean, was < 320 nm. At presentation, 95% had microscopic haematuria, 12% macroscopic haematuria, 14% loin pain, 28% proteinuria, and 14% hypertension. There was no difference in GBM width between the sexes (male 258 nm vs. female 251 nm) but there was a significant negative correlation between age and GBM width (r = -0.53, p < 0.001), with older patients having the thinnest membranes. Twenty six patients had ultrathin GBM (< 270 nm), of whom 54% had 3+ haematuria vs. 12% of the group with BM > 270 nm (p < 0.01). In the ultrathin group, 71% had loss of anionic charge from the GBM, vs. 17% in those with membranes which were thin but > 270 nm (p < 0.05). Proteinuria occurred more frequently in those with GBM > 270 nm, 65% vs. 8% in the ultrathin group (p < 0.01). Thin GBM were associated with a benign prognosis, as after a mean follow-up of 85 months (48-140), there was no significant change in either serum creatinine or mean arterial blood pressure. Patients with ultrathin GBM had greater loss of GBM anionic charge, which might result in both an alteration of flow characteristics within the glomerular capillaries and also increased fragility of the glomerular basement membrane with likelihood of rupture and resultant macroscopic haematuria.

Cationic gold staining of glomerular anionic sites in archived tissue, reprocessed from paraffin wax into LR gold resin.

Glomerular capillary wall anionic sites have been demonstrated by cationic gold staining of archived renal biopsy tissue (up to 10 years old), obtained from six patients, originally embedded in paraffin wax, and subsequently reprocessed into LR gold resin. The staining patterns at pH 2.5 and pH 7.0, demonstrating different glomerular basement membrane (GBM) anionic constituents, were compared in three patients from whom tissue directly processed into LR gold and reprocessed tissue was available. Ultrastructural preservation was poorer and shrinkage artefact greater in paraformaldehyde-lysine periodate (PLP) as opposed to formol saline-fixed reprocessed tissue. However, GBM anionic site expression was well preserved, or even enhanced (lamina rara externa, pH 7.0) in reprocessed tissue, using either fixative. Although it may not be possible to compare subtle changes in anionic site distribution in variously fixed and processed tissues, due to these artefacts, the technique enables retrospective study of charge status in archived material from disease groups in which there are distinct anionic site aberrations.

Preparation and use of the poly-L-lysine-gold probe: a differential marker of glomerular anionic sites.

The conditions required for the production of a polylysine-coated gold (PL-G) complex, which shows optimal sensitivity for the demonstration of tissue anionic sites, expressed under different conditions of pH have been investigated. Problems encountered with this complex have been compared with those found with other methods of conjugation of polylysine to colloidal gold. The performance of a bovine serum albumin (BSA)-stabilized PL-G complex was examined against other PL-G conjugates, including complexes that are commercially available, for the detection of heterogeneous glomerular anionic site populations, expressed at pH 2.5 and pH 7.0.

Detection of glomerular anionic sites in post-embedded ultra-thin sections using cationic colloidal gold.

We detected glomerular anionic sites in fixed, LR Gold-embedded ultra-thin tissue sections using cationic colloidal gold. Manual and computer-assisted quantitation were compared, and the influence of pH and glycosaminoglycan-degrading enzymes on site expression was examined. Both quantitation methods produced similar results. Alteration of pH 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 lamina rara externa were stained. At pH 3.0 and above, glycocalyx was unstained but intracellular and nuclear staining was present; glomerular basement membrane (GBM) and mesangial matrix sites were abundant. After chondroitinase ABC or hyaluronidase digestion, GBM staining was eliminated at pH 2.0 and reduced at pH 7.0 (p less than 0.001), suggesting that degraded sites are associated with chondroitin sulfate or hyaluronic acid. By contrast, prolonged heparitinase I digestion was ineffective at either pH. Digestion of purified substrates revealed crossreactivity of heparitinase towards chondroitin sulfate and of chondroitinase towards hyaluronic acid. Since tissue sites were reduced by chondroitinase but not heparitinase, we suggest that degradation is due to hyaluronidase activity of chondroitinase and the anionic sites are associated with hyaluronic acid. However, the influence of pH indicates that lamina rara externa sites are structurally distinct from other GBM anionic sites.

Cationic colloidal gold--a novel marker for the demonstration of glomerular polyanion status in routine renal biopsies.

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.

Immunogold-silver staining of mesangial antigen in lowicryl K4M- and LR gold-embedded renal tissue using epipolarization microscopy.

We examined the localization of a glomerular mesangial antigen with a Thy-1.1 monoclonal antibody by epipolarization microscopy (EPI) of silver-enhanced, immunogold-stained renal tissue embedded in LR Gold and Lowicryl K4M, and compared the attributes of these hydrophilic resins. Antigen was well preserved in tissue embedded in both resins. LR Gold-embedded tissue demonstrated excellent immunostaining properties, sectioned more easily, and showed better durability during staining than K4M. Lowicryl K4M-embedded tissue, however, displayed a phenomenon of self-illumination when counterstained with eosin which was not seen with LR Gold. This enabled immunostaining to be precisely related to tissue morphology without the necessity of simultaneous transillumination, which can be problematic when used in combination with EPI because of reflection of incident illumination from sub-stage optical surfaces.

Spontaneous glomerular immunoglobulin deposition in young Sprague-Dawley rats.

The frequency, age-onset and distribution of spontaneously deposited immunoglobulins (Igs) in glomeruli of Sprague-Dawley rats has been investigated. Groups of rats (n = 10) were examined at 4-7 day intervals from birth (presuckling) until 30 days of age. Findings were compared with circulating immunoglobulin concentrations in each age group. Immunoglobulins were undetectable in immature kidneys of newborn rats. However, as early as 5 days, scanty IgA and IgM deposits were observed predominantly in mesangial areas of mature glomeruli, corresponding to low circulating concentrations of these immunoglobulins. By contrast, glomerular IgG deposits were not observed until 21 days, despite relatively high concentrations of circulating maternal IgG from birth. Mesangial deposition of immunoglobulins increased with age. Absence of complement C3c or electron dense deposits associated with this mesangial localization suggests that immunoglobulins were not deposited as immune complexes. Accumulation of non-phlogogenic immunoglobulins in the mesangium of normal rats supports the concept that the mesangium is constantly perfused by circulating macromolecules and filtration residues. The results indicate problems of interpretation of the significance of endogenous immunoglobulin deposition in models of experimental glomerulonephritis, even in studies involving weanling rats.

Glomerular inflammation: a consequence of sequential challenge of the rat mesangium with particulate macromolecules.

Imposil pretreatment (50 mg/100 g bodyweight) of male Sprague-Dawley rate (100-150 g), 24-48 h before an albumin-coated latex challenge resulted 24 h later in markedly reduced mesangial endocytosis of latex, compared with control animals receiving latex-albumin alone. This reduced capacity to ingest latex was associated with increased glomerular infiltration by Ia+ macrophages and mesangial localisation if immunoglobulins and complement C3c. In sharp contrast, pretreatment with albumin-coated latex had little effect on the subsequent mesangial uptake of Imposil. In this reverse situation macrophage infiltration was minimal, immunoglobulin deposition was similar to that in untreated control animals, and C3c was not detectable. Similarly, extending the interval between Imposil pretreatment and albumin-coated latex challenge up to 144h resulted in only minor infiltration by Ia+ macrophages and immunoglobulin localisation without accompanying C3c deposition, even though mesangial latex uptake was still limited. These observations demonstrate the consequences of secondary challenge following mesangial impairment due to a predisposing insult. Glomerular inflammation was dependent upon the timing and nature of the sequential challenge. The results support the concept that mesangial impairment following an initial insult may be a major factor in the predisposition to some forms of human glomerular inflammation.

Preparation, purification and analysis of BSA-coated latex particles for in vivo studies. Non-ionic adsorbance of unbound protein ligand.

A procedure for the preparation, purification and analysis of small, BSA-coated carboxylate-modified latex (CML) particles, suitable for use in in vivo studies has been developed. Following conjugation, uptake of unbound BSA by Amberlite XAD-8 non-ionic adsorbent beads has been shown to be an effective method by which unbound protein ligands may be removed from coated latex preparations.

Uptake and disposal of BSA-coated latex particles by the rat mesangium: reaction with subsequently administered heterologous antiserum.

Mesangial uptake and disposal of antigen-coated latex particles and the ability of subsequently injected antibody to maintain complexed antigen in the rat mesangium has been investigated. Carboxylate-modified latex particles, coated with bovine albumin (BSA) were injected i.v. to 36 Wistar rats. Twenty-two rats (group 1) were not treated further. Fourteen rats (group 2) received rabbit anti-BSA antiserum i.v. and i.p. 24 h later. Control groups were injected with uncoated, unmodified latex particles or soluble BSA with and without subsequent antibody administration. Latex was present in the mesangial matrix of rats in group 1 at 1 h in association with a diffuse mesangial distribution of BSA. At 24 h, BSA staining was markedly reduced and extracellular latex was no longer observed. Intracellular latex aggregates were present in experimental and control groups at 24 h-14 days in cytoplasmic vacuoles of hypertrophic mesangium which showed minor infiltration by macrophage-like cells. Progressive removal of latex aggregates coincided with declining mesangial reactivity. Rapid disappearance of antigen apparently results from local degradation of tracer in the mesangium. Antibody administration preserves BSA in the mesangium due to immune complex formation and is associated with retention of ingested latex by mesangial cells. However, efficient disposal of glomerular immune deposits by the mesangium appears to minimize infiltration by monocytes and prevents aggravation of glomerular inflammation.

Persistence of inert macromolecules (Imposil) in the rat mesangium and glomerular functional disturbance.

Imposil iron-dextran is an inert tracer that has been used to study mesangial uptake and clearance of macromolecular material from the glomerular circulation. Such a tracer may be a useful marker of altered mesangial function in animals with some forms of glomerulonephritis. We have studied mesangial handling of intravenously injected Imposil (50 mg/100 g body weight) in normal rats by light, immunofluorescence and electron microscopy for up to 3 months. Mesangial cell uptake was maximal at 48-54 h. Extrusion and drainage of tracer to the vascular pole and distal tubule was evident at 3 days but iron was still present in mesangial cells at 3 months. Possible functional renal impairment resulting from persistent mesangially sequestered tracer was examined by measuring daily urine protein and iron excretion. A possible relationship between failure of mesangial cells to eliminate inert tracer and increasing glomerular permeability is demonstrated, suggesting that Imposil and similar inert macromolecules cannot be used for long-term studies of mesangial function.