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The contractile response to bradykinin (BK), measured by the reduction of the planar surface area, was studied in glomeruli and mesangial cells (MC) isolated from diabetic rats (D) one week after diabetes induction with injection of streptozotocin (STZ; 60 mg kg-1, i.p.). Results were compared with age and weight-matched untreated rats (N) and were expressed by two parameters of cell activity, the mean maximum contraction (MMC) and the proportion of contractile cells (PCC). Glomerular and mesangial contraction were found to be clearly reduced in diabetic rats in response to 100 nM BK. The lower contractile response was associated with a decrease of both glomerular calcium uptake and mesangial cell intracellular calcium mobilization. The fact that cell pretreatment with two protein kinase C (PKC) inhibitors, phorbol 12-13 myristate acetate and calphostin, lowered normal cell contraction at the level of that found in diabetic MC without any significant effect in the latter, suggests the involvement of a PKC pathway, perhaps by a decrease of activatable PKC in diabetes. In addition, our results led to the first description of a possible role of the kallikrein-kinin system in the early glomerular hemodynamic changes occurring in diabetes. Insulin (1-200 nM) increased the contractile response of cultured diabetic cells (MMC), and in this case, it also increased the PCC. It must be stressed that the effect of 1 nM insulin on the former (88% increase) was very much smaller than its effect on the latter (103% increase). The combination of the two parameters (contraction index, CI) provided a realistic evaluation of the contractile capacities of the cell population of the cultures as a whole. The differences in this index between normal and diabetic cell populations, in the absence or presence of insulin, were strictly parallel to those found in intact glomeruli. Finally, our results further confirm (Ouardani et al., Biol. Cell 86, 127, (1996)) the limit of the first five cell passages within which cultured MC can be reasonably used for the study of contractile abnormalities occurring in the early steps of diabetic state.

To improve understanding of human profilaggrin processing to filaggrin, we produced seven monoclonal antibodies against epidermal filaggrin (AHF1-7). They were characterized on human epidermis by indirect immunofluorescence, immunogold labeling, and immunoblotting and found to be directed against seven different epitopes of (pro)filaggrin. AHF1-5 labeled the keratohyalin granules and the fibrous matrix of the lower corneocytes, and recognized filaggrin and profilaggrin. AHF6 also labeled the keratohyalin granules and the corneocyte matrix, but only recognized filaggrin. In addition to this reactivity within the upper epidermis, AHF4-6 stained the cytoplasm of the basal cells, and cross-reactivity of AHF5 and AHF6 with cytokeratin K14 was revealed on immunoblots. It is interesting that AHF7 recognized filaggrin, but not profilaggrin, and labeled only the corneocyte matrix and not the keratohyalin granules. This indicates that filaggrin and cytokeratins share several antigenic determinants and that filaggrin bears at least one epitope absent from its precursor. The original series of monoclonal antibodies described here appears to be a powerful tool for studying human profilaggrin processing in normal conditions and in the keratinization disorders in which processing is altered.

The effect of bradykinin (BK) on the contraction of rat mesangial cells (MC) was compared with that of various vasoactive agents. BK induced a dose-dependent contraction [one-half maximal effective dose (ED50) = 50 nM] inhibited by the B2 antagonist, HOE-140 (ED50 = 10 nM). BK-induced MC contraction was independent of extracellular calcium and was reduced by inhibition of protein kinase C (PKC). Neomycin completely prevented the increase in intracellular calcium and the formation of inositol 1,4,5-trisphosphate induced by BK but only reduced cell contraction. Inhibition of prostaglandin (PG) formation and administration of the endoperoxide antagonist SQ-27427 also partly decreased the effect of BK. Interestingly, only the addition of both neomycin and mepacrine resulted in a complete inhibition of cell contraction. These results suggest that BK, via a B2-kinin receptor, induces contraction of MC through two distinct mechanisms, one associated to the phospholipase C pathway and subsequent activation of PKC and the second one dependent on PG formation. These in vitro effects may be relevant in explaining the effects of BK and converting enzyme inhibitors on glomerular hemodynamics.

Objectives: To assess the diagnostic value for rheumatoid arthritis (RA), of an immunoblotting assay based on the rat oesophagus epithelium antigens recognised by the so-called 'antikeratin antibodies' ('AKA'), antigens that have been identified as three non-cytokeratin proteins (referred to as A, B and C proteins)., Methods: After polyacrylamide gel electrophoresis in non-denaturing conditions and electrotransfer of an epithelial extract, the immunoreactivities to the A, B and C proteins of a series of serum samples from 88 patients with RA and 100 patients with non-rheumatoid rheumatic diseases, were semiquantitatively evaluated., Results: A total of 81.8% of RA serum samples recognised the three proteins, while 91% of non-RA serum samples only weakly recognised the A and B proteins but not the C protein. Only in the group of RA patients, were the titres of the antibodies to the A, B and C proteins found to be significantly correlated with each other and with the titres of 'AKA' detected by the standard indirect immunofluorescence (IIF) method. For a diagnostic specificity of 99%, the diagnostic sensitivities of the detection of the A and B proteins were 50% and 43.2%, respectively, when those of the detection of 'AKA' by IIF and of IgM-rheumatoid factor by enzyme-linked immunosorbent assay were 42% and 54%, respectively. In contrast, at a same specificity of 99%, the diagnostic sensitivity of the detection of the C protein was significantly higher with a value of 70.5%., Conclusion: This immunoblotting assay which is the first immunochemical method proposed for the detection of 'AKA, should be validated on larger series of patients but can already be considered as a very powerful test for the serological diagnosis of RA.

We investigated the effect of two oral (p.o.) doses of cicletanine (5 and 30 mg/kg/day) for 4 weeks on urinary excretion (UKE), renal concentration (RKC) of kallikrein, and prostaglandin E2 (PGE2) and 6-keto-PGF1 alpha urinary excretion of stroke-prone (SP) spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) rats submitted to a high sodium intake (1%). Both doses of cicletanine induced a significant antihypertensive effect in treated SHR as compared with hypertensive untreated controls (HC). After 4-week treatment, a significant difference in mortality was observed between normotensive controls (NC) (0%) and HC (84%). Both doses of cicletanine reduced the mortality of hypertensive animals (8% SHR with 5 mg and 24% SHR with 30 mg vs. 84% in HC). Whereas UKE and RKC were decreased in HC during the progression of untreated hypertension from week 1 to week 4, both doses of cicletanine administration significantly prevented this decrease. Consistently with maintenance of UKE during the course of hypertension, the level of tissue kallikrein was higher in hypertensive cicletanine-treated than in untreated SHR. This increased RKC was associated with a significantly higher rate of kallikrein biosynthesis. The increased level of the urinary excretion and tissue concentration of PGE2 and 6-keto-PGF1 alpha in cicletanine-treated SHR as compared with untreated animals was also of interest. This protective effect on PG excretion correlated with that on kallikrein excretion. The results confirm the efficiency of cicletatine as an antihypertensive treatment. The antihypertensive action includes protective effects on potential vasodepressor kallikrein-kinin and prostaglandin systems.(ABSTRACT TRUNCATED AT 250 WORDS)

To extend our recent observations of the possible downregulation of glomerular B2-kinin-binding sites, we investigated density (Bmax) of bradykinin (BK)-binding sites in glomerular membranes of both the clipped (C) and nonclipped (NC) kidneys of two-kidney, one-clip (2K-1C) Goldblatt hypertensive rats, in relation to tissue kallikrein activity and glomerular three-dimensional structure. Compared with the Bmax of sham-operated (SO) kidney (31.8 +/- 7 fmol/mg protein), a significant increase in Bmax was observed in glomeruli of both kidneys in hypertensive rats, the Bmax being higher in glomeruli of NC than in C kidneys (98 +/- 11 vs. 59 +/- 12 fmol/mg protein). NC kidney compensatory hypertrophy was expressed by an increase in glomerular diameter, surface area, and volume. When expressed per unit of area or volume, Bmax in NC kidneys remained significantly higher than in both C and SO kidneys. Increased Bmax in both kidneys of 2K-1C rats was associated with a decreased intrarenal level of kallikrein. We also examined prostaglandin (PG) E2 release by isolated glomeruli from SO, C, and NC kidneys as a possible biological effect induced by BK. Whereas C kidney released more PGE2 than NC kidney under basal conditions, addition of BK (10 nM) induced greater PGE2 production in NC kidney consistent with the difference in Bmax between C and NC kidneys. These results suggest a possible downregulation of glomerular B2-binding sites by bradykinin, which may explain the difference between SO and C kidneys.(ABSTRACT TRUNCATED AT 250 WORDS)

The authors report the results of immunofluorescence (IF) studies of 17 cases of "non-idiopathic" renal biopsy-proven amyloidosis and 18 cases of various nephropathies and normal kidneys (as controls), investigated by IF by simultaneous use of antisera against routine IgG, IgM, IgA, C3, C4, Clq, beta-lipoprotein, albumin, and fibrinogen. Antisera against kappa and lambda light chains and amyloid A and amyloid P components were also used. Six of the 17 cases of amyloidosis were associated with immunocyte dyscrasia, and 11 were cases of reactive systemic amyloidosis associated with chronic infections or inflammatory and neoplastic disorders. In amyloidosis, IF deposits appeared for all antisera as homogeneous staining of mesangial nodules, and, more rarely, there was staining along the glomerular basement membranes. Overall immunoglobulins and C3 were present in 11 cases (64 per cent). Kappa and lambda light chains were demonstrated in 14 (82 per cent) and 12 (70 per cent) cases, respectively. In immunocyte dyscrasia associated with amyloidosis, immunoglobulin and light-chain deposits corresponding to a paraprotein abnormality were demonstrated in glomeruli and in tubular casts. Amyloid P component was always present in glomeruli with a bright and characteristic fluorescence, and it was frequently observed in arterioles. Amyloid A component was observed in six cases of reactive systemic amyloidosis but also in one case of immunocyte dyscrasia with amyloidosis. In view of the diversity of amyloid fibril types and their chemical nature, IF studies confirm the presence of different constituents but do not warrant any conclusion concerning the pathogenesis of this disease.

Two immune sera have been raised in sheep against rabbit glomerular and tubular basement membranes, respectively (GBM and TBM). Their specificity was investigated by immunoabsorption and radioimmunoassay, and their nephrotoxicity was studied by injecting them into New Zealand rabbits. Both antisera reacted in vitro collagen and noncollagen-related glycopeptides. However, while the anti-GBM antiserum reacted preponderantly with GBM and TBM collagen which probably represent the antigen(s) shared by the two basement membranes, the anti-TBM antiserum was directed preferentially against the noncollagenous component of rabbit TBM. The anti-GBM antiserum induced in rabbits a diffuse extracapillary glomerulonephritis with linear fixation of autologous IgG and C3 along the GBM. The anti-TBM antiserum also induced an extracapillary glomerulonephritis but in addition a focal tubular atrophy was seen and autologous IgG and C3 were fixed along both GBM and TBM and glucosuria frequently occurred. These results suggested that the glomerular lesions induced by the two antisera were due to anticollagen antibodies while the tubulointerstitial lesions were due to antibodies directed against the noncollagenous portion of TBM.

The localization of beta 2-microglobulin (beta 2m) was studied in renal biopsies from 18 patients with pathological transplanted kidney using immunofluorescence and electron-immunohistochemical techniques; the renal biopsies of 4 cases with normal kidneys were used as controls. Using immunofluorescence, beta 2m was not observed in the normal kidneys, beta 2m was found in the glomeruli (7 cases) and the tubular epithelium (16 cases) of the transplanted kidneys. Using immunoelectron microscopy, some labelling of the normal kidneys was observed mainly along the cell coat of foot processes and in tubular-epithelial lysosomes. In the glomeruli of transplanted kidneys, particularly in cases of acute or chronic rejection, beta 2m was most frequently localized on the outer layer of the basement membrane and along the cell coat of foot processes. The brush border of the proximal tubules and the lysosomal structures were intensely labelled. Although immunoelectron-microscopy studies are unable to discriminate between the localization of beta 2m in normal and transplanted kidneys, these findings nevertheless suggest the glomerular filtration of beta 2m and its metabolism in the tubular epithelium.

The localization of amyloid P-component (AP) staining was studied by immunofluorescence in renal biopsies from 106 patients with various nephropathies and from 3 patients with normal kidneys. Linear staining was observed along the glomerular basement membranes (GBM) of normal kidneys. In amyloidosis, AP was always present in the glomeruli. In arteriolar walls, AP was present in numerous cases with varied intensity. No fixation was observed along tubular basement membranes. The possible modification of the permeability of GBM, related to a possible modification of the electrical charge of the filtration barrier, can be supposed.

This preliminary study concerns murine monoclonal antibodies (Mabs) against tubular basement membranes (TBM) of human kidney which were produced by the classical hybridization technique. One of these Mabs (PP8-1) specifically recognized an antigen present in the tubular basement membranes of various portions of tubule and in the Bowman's capsule. By immunocytochemical techniques, this Mab was used to study the presence and the distribution of the related antigen in urinary sediment from 12 normal healthy subjects. This TBM and Bowman's capsule antigen was demonstrated along hyaline urinary cast surface and, in some cases, in free extracellular fibrillar structures morphologically unrelated to the casts. This suggests that, in some normal conditions, catabolism of TBM and/or Bowman's capsule could release material in urine, as it has been described for glomerular basement membrane (GBM) constituents. Such an immunocytochemical approach could be useful to detect components from tubular renal basement membranes in urinary sediment during pathological conditions.

A method allowing simultaneous isolation of Rabbit alomerular and tubular basement membranes has been perfected. Rabbit tubular basement membrane has also been obtained by tryptic digestion of the renal cortex. Electron microscopic observation and biochemical analysis show that the contamination of the membrane preparations by cellular components is negligible.

A nephropathy affecting both tubules and glomeruli has been induced in Rabbits by the injection of an heterologous immune serum directed against Rabbit tubular basement membrane. It differs from experimental antiglomerular basement membrane nephropathy by the fixation of antibodies and C3 on tubular basement membrane and by the occurrence of glucosuria.

Renal tissue from 185 patients with various nephropathies were studied by immunofluorescence, in order to look for the frequency and potential predominance of kappa or lambda light chain glomerular deposits. Four normal renal biopsies were used as controls. An overall study shows that light chains were present in glomeruli in 136 out of 185 cases; kappa light chain deposits were more frequent than lambda light chain deposits (73.5% and 64.3% respectively). An analytical study shows that this was not observed in all nephropathies studied. In mesangial IgA nephropathy, lambda light chain deposits were seen in 81% of cases (29 out of 37) and kappa light chain deposits were observed in 78% (30 out of 37 cases). In lupus nephritis, lambda light chain deposits were present in 13 out of 14 cases (92.8%) whereas kappa light chain deposits were demonstrated in 12 cases (85.7%). In other nephropathies such as membranous, endocapillary proliferative and amyloid nephritis, kappa was the predominant light chain observed in glomeruli or was present in the same number of cases as lambda light chain (mesangiocapillary glomerulonephritis). These findings show that in certain nephritides, for example IgA nephropathy and lupus nephritis, IgA and IgG deposits are mainly composed of lambda light chain in contrast with the normal kappa: lambda ratio in human serum of 2:1.

Renal biopsy specimens obtained from patients with various chronic nephropathies were studied by immunofluorescence (IF), electron and light microscopy. Of 49 biopsies studied by IF, 22 had granular deposits of IgG and/or C3 along the basement membranes of proximal tubules while 14 had linear and 13 both linear and granular deposits. The possible importance of immunologically mediated renal tubular lesions in chronic nephropathies is discussed.

In 18 patients who received renal allografts, tubulo-interstitial lesions were studied, according to the types of rejection or the possible recurrence of primary disease. Immunofluorescent (IF) investigation revealed tubular basement membrane (TBM) deposits of IgG and/or C3 and sometimes other components of Complement. The pattern of these deposits was: "linear" in 3 cases, "granular" in 9 cases and "atypical" with linear and granular segments in 6 cases. The IF study of own kidneys of 3 patients, before the transplantation, revealed no TBM deposits. TBM deposits were accompanied by arterial wall deposits of IgM and/or C3 and sometimes C5 in 17 cases. Electron-microscopy study of renal tubules showed thickening, lamellation and/or deposits along the tubular basement membrane. The immunopathogenesis of these deposits was discussed.