Eduard Mirapeix

TRPC6 mutational analysis in a large cohort of patients with focal segmental glomerulosclerosis

BACKGROUND Mutations in the TRPC6 gene have been reported in six families with adult-onset (17-57 years) autosomal dominant focal segmental glomerulosclerosis (FSGS). Electrophysiology studies confirmed augmented calcium influx only in three of these six TRPC6 mutations. To date, the role of TRPC6 in childhood and adulthood non-familial forms is unknown. METHODS TRPC6 mutation analysis was performed by direct sequencing in 130 Spanish patients from 115 unrelated families with FSGS. An in silico scoring matrix was developed to evaluate the pathogenicity of amino acid substitutions, by using the bio-physical and bio-chemical differences between wild-type and mutant amino acid, the evolutionary conservation of the amino acid residue in orthologues, homologues and defined domains, with the addition of contextual information. RESULTS Three new missense substitutions were identified in two clinically non-familial cases and in one familial case. The analysis by means of this scoring system allowed us to classify these variants as likely pathogenic mutations. One of them was detected in a female patient with unusual clinical features: mesangial proliferative FSGS in childhood (7 years) and partial response to immunosupressive therapy (CsA + MMF). Asymptomatic carriers of this likely mutation were found within her family. CONCLUSIONS We describe for the first time TRPC6 mutations in children and adults with non-familial FSGS. It seems that TRPC6 is a gene with a very variable penetrance that may contribute to glomerular diseases in a multi-hit setting.

Anti-myeloperoxidase autoantibodies in patients with necrotizing glomerular and alveolar capillaritis.

We conducted a prospective study of 651 Mediterranean patients from Catalonia (Spain) with well-defined forms of systemic vasculitis, connective tissue diseases, and renal and pulmonary disorders to determine the prevalence and clinical value of antineutrophil cytoplasmic autoantibodies (ANCA) with myeloperoxidase (MPO) specificity (MPO-ANCA). ANCA were first tested by indirect immunofluorescence on ethanol-fixed neutrophils. When a positive result was obtained, then MPO-ANCA were identified by performing the immunofluorescence assay again on neutrophils from a voluntary donor known to have a complete and selective deficiency of MPO. This disorder was detected by automated flow cytochemistry with the Technicon system and was further verified by cytochemical and biochemical studies. We detected MPO-ANCA in 61 of 70 (87%) patients with a perinuclear pattern (p-ANCA), but in none of 25 with a cytoplasmic pattern (c-ANCA). These results were corroborated by enzyme-linked immunosorbent assay (ELISA) using human purified MPO as a substrate. On immunofluorescence microscopy, all patients with MPO-ANCA were found to have a typical and restrictive immunostaining pattern. In our study, while c-ANCA were mainly found in patients with biopsy-proven Wegeners granulomatosis, MPO-ANCA identified those with idiopathic and polyarteritis nodosa-associated necrotizing and crescentic glomerulonephritis. In addition, pulmonary hemorrhage with necrotizing alveolar capillaritis as the main morphologic substrate occurred frequently among patients with MPO-ANCA, including three affected by polyarteritis nodosa and three who had pulmonary hemorrhage as the only clinical finding. On the other hand, these antibodies could be also detected in 30% of patients with a proven diagnosis of anti-glomerular basement membrane (GBM) disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term outcome of antineutrophil cytoplasmic antibody-associated small vessel vasculitis after renal transplantation

The survival after renal transplantation of patients with antineutrophil cytoplasmic antibody (ANCA)‐associated to systemic vasculitis is as good as in other diseases, although most of the reports are based on small numbers of patients. Furthermore, it is not known whether comorbidities (cardiovascular [CV] disease and cancer) are more frequent than in general population. We report our experience and the analysis of the published data on this topic. The outcome after transplantation in 49 patients with ANCA‐associated small vessel vasculitis was compared with a control group. The relapse rate of vasculitis was 0.01 per patient per year. Comparison with the control patients revealed no difference in long‐term outcome, CV mortality or incidence of malignancies. In the published literature, patients with ANCA at transplantation and with Wegeners granulomatosis are at greater risk of relapse. Taking our own results together with the review of the literature, we conclude that patient and graft survival rates compare favorably with those in control group that the recurrence rate is very low and that there is no increase in the incidence of cancer or in CV mortality. Patients with ANCA at transplantation and with Wegeners granulomatosis have a higher relapse rate.

Vasculitis syndromes: LAMP-2 illuminates pathogenesis of ANCA glomerulonephritis.

The discovery that antibodies to a bacterial antigen can cross-react with a mammalian protein to cause pauci-immune necrotizing and crescentic glomerulonephritis opens up new possibilities for the diagnosis and treatment of this condition.

Cellular Immunity Analysis Using Monoclonal Antibodies in Human Glomerulonephritis

Monoclonal antibodies against class II antigens of the human major histocompatibility complex (MHC) (Edu 1), von Willebrand factor-related antigen marker of endothelial cell, T cells (Cris 1), helper/inducer T cells (T4) and cytotoxic/suppressor T cells (T8) by indirect immunofluorescence, and stain for nonspecific esterase characterizing monocytes-macrophages (Mo-Ma) were applied in 64 renal biopsies--54 glomerulonephritis (GN), 10 non-GN- and in 14 normal kidneys. Class II antigens were expressed on the endothelium of renal microvasculature in all specimens. Intraglomerular T cells and Mo-Ma were only present in GN. Mo-Ma appeared associated with endo- and extracapillary proliferation (Xc2 = 4.68; p less than 0.05), C3 (X2 = 4.21; p less than 0.05), and fibrinogen (X2 = 3.84; p less than 0.05) deposition; and those were most numerous in biopsies with intraglomerular T cells. Interstitial MHC-class II+ cells (Xc2 = 5.5; p less than 0.02), T cells (F = 3.37; p less than 0.005) and Mo-Ma (F = 2.45; p less than 0.05) were significantly higher in GN with endo- or extracapillary proliferation than in the remaining. In GN, correlations were seen between T cells and MHC-class II+ cells (r = 0.63; p less than 0.001), and Mo-Ma (r = 0.38; p less than 0.02), infiltrating the interstitium. Our results suggest that both humoral and cellular immunity contribute to macrophage glomerular infiltration in the human GN. Mononuclear cells, and no intrinsic renal cells, would be implicated in the cellular immune interactions in situ.

DIALYSIS MEMBRANES INHIBIT SYNTHESIS AND RELEASE OF BETA 2-MICROGLOBULIN IN LYMPHOCYTE CULTURE

J.M. Campistol Plana, Arthritis Center (K-5), Boston University School of Medicine, 7. 71 East Concord Street, Boston, MA 02118 (USA) Dear Sir, We have read with interest the article of Paczek et al. [1] about the inhibitory effect of dialysis membranes on ß2-microglobulin synthesis and release from human lymphocyte culture. Nowadays, dialysis-amyloidosis represents a frequent and invalidating complication of patients with chronic renal failure on hemodialysis treatment, specially after long-term period. The pathogenesis remains unknown and the significance of ß2-microglobu-lin serum levels and intradialysis production also remain questioned [2]. In a similar study to that of Paczek et al., trying to determine the role of dialysis membranes on ß2-microglobulin synthesis, we obtained close results, confirming the inhibitory effect of dialysis membranes on ß2-microglobulin release from lymphocyte culture. Twenty-four patients (mean age 52 ± 11 years) affected with chronic renal failure and on hemodialysis treatment for a mean time of 5.7 ± 3.6 years, and a control group of 6 subjects with normal renal function and a mean age of 39 + 5 years old, took part in the study. ß2Microglobulin was determined in the supernatant using commercially available EIA(Phadezym ß2-Microtest; Pharmacia Diagnostics) with a sensitivity between 3.3 and 500 ng/ml. 500 ∏) 400 ■ ω 300 o 200 100 AN-69* Basal Cuprophane Cuprophane* AN-69 Fig. 1. Inhibitory effect on ß2-microglobulin synthesis and release by dialysis membranes.* Dialysis membranes sterilized with gamma rays. Peripheral blood lymphocyte cultures were performed with conventional methodology [3] during an incubation period of 9 days. Two different short tubes of dialysis membranes were incubated in lymphocyte cultures: cuprophane (ST-15; Travenol) and polyacrylonitrile (AN-69; Hospal): Dialysis membranes were sterilized in two different ways, i.e., with ethylene oxide and with

Cellular Infiltrate in Renal Graft Rejection: T Lymphocyte Subsets Detected by Monoclonal Antibodies

We have examined the interstitial cellular infiltrate using monoclonal antibodies against T cells (Cris 1), helper/inducer T cells (OKT4) and suppressor/cytotoxic T cells (OKT8) by indirect immunofluorescence in renal biopsies taken from 14 transplanted patients during clinical episodes suggestive of acute (n = 9), chronic (n = 2) and no rejection (n = 3). Infiltrating T cells and T cell subsets were found to be significantly increased during all types of rejection (n = 11) as compared to no rejection (n = 3). Two types of biopsies could be distinguished according to the predominance of T cell subsets. In some biopsies (n = 6), OKT8+ cells were significantly more numerous that OKT4+ cells. In the remaining biopsies (n = 5), OKT4+ cells were more common that OKT8+ cells, the OKT4/OKT8 ratio being significantly higher. No association was observed between HLA mismatch and predominating T cell subset, neither for type nor outcome of graft rejection. Our results suggest that the OKT4+ cells may play a more important role than previously reported in renal graft rejection.

DR expression on vascular endothelial cells in normal human kidney.

P. Arrizabalaga, MD, Servicio de Nefrología, Hospital Clínico, c/Casanova, 143, E-Barcelona 36 (Spain) Dear Sir, Human HLA-DR antigens are expressed on cytoplas-mic membrane of cells associated with immunological activity [1]. Moreover, the HLA-DR expression can be induced on other cells which are normally negative for HLA-DR molecules. Thus, the thyroid follicular cells bear these antigens when cultured with mitogens [2]. The umbilical vein endothelial cells express HLA-DR when cultured with phytohemagglutinin [3] or co-cultured with activated T cells [4]. Häyry et al. [5] observed HLA-DR antigens on the dispersed kidney vascular endothelial cells. In addition, morphological studies on tissue sections suggest that renal vascular endothelium appears to express HLA-DR antigens [6–8]. We have observed, using immunofluorescence (IF) microscopy and a nucleic acid counterstain with ethidium bromide (EB), that in the normal human kidney these antigens are localized on the vascular endothelium around cellular structures. We have used a mouse monoclonal antibody directed against a monomorphic determinant of HLA-DR (Edu-1) described elsewhere [9, 10]. Normal renal tissue from 2 biopsy and 9 necropsy specimens was tested for HLA-DR antigens by indirect IF technique. A nucleic acid stain with EB was used for cellular localization. Tonsil sections were used as positive control. Monoclonal antibody to non-HLA-DR antigens (Cris-1) [9,11] and ascitic fluid, obtained intraperitoneally of Balb/c NSA myeloma line were used as negative controls. A constant pattern of HLA-DR antigens was observed in all the specimens. Heavy IF staining was identified in the renal interstitium and in the glomerular capillary walls. Moreover, bright staining was observed in the mesan-gium. EB counterstaining showed HLA-DR antigens around endothelial cells in glomerular capillaries (fig. la) and probably vascular endothelial cells in intertubular capillaries (fig. lb). The considerable amount of HLA-DR antigens observed in capillaries of human normal kidney suggest at least two biological implications. First, the vulnerability of the microcirculation of transplanted kidney to circulating antibodies with specificity for HLA-DR antigens

Kinetic analysis of changes in T- and B-lymphocytes after anti-CD20 treatment in renal pathology

INTRODUCTION The main objective of this study is to describe qualitatively and quantitatively the different immune lymphocyte phenotypes of patients with renal disease after treatment with anti-CD20. MATERIAL AND METHODS Two cohorts of transplanted and autoimmune kidney patients were compared: (1) Those who began treatment with Rituximab, matched (for sex, age and general clinical parameters) with (2) Non-treated control kidney patients. Different analyses were performed: (A) B-lymphocyte subpopulations; (B) T-cell subpopulations; (C) serum levels of BAFF, APRIL, Rituximab and anti-Rituximab; (D) rs396991 polymorphism of CD16a and at different time points for each type of analysis: (i) at baseline, (ii) day 15, (iii) at three and (iv) six months post-antiCD20. RESULTS (A) A depletion of all B cell subsets analysed was observed preferentially decreasing the CD40+memory B-cells, switched memory cells and plasmablasts. (B) A significant decreased percentage of CD4+T-lymphocytes was observed. A significant decrease of the percentage of memory T-cells and an increase in naïve T-cells was also observed