Functional Characterization and Pharmacological Evaluation of a Novel GLA Missense Mutation Found in a Severely Affected Fabry Disease Family.

BACKGROUND: Fabry disease (FD) is a rare X-linked storage disorder resulting from the deficient activity of the lysosomal hydrolase α-galactosidase A (α-Gal A). Here we describe a 23-year-old man with FD possessing a novel mutation in the GLA gene, the evaluation of his family, and the functional characterization of the novel variant. METHODS: Two generations of a family were screened for FD by clinical symptoms and low enzymatic activity. This step was followed by DNA sequencing that showed a novel GLA missense mutation. To confirm the pathogenicity potential of the mutation, we employed site-directed polymerase chain reaction mutagenesis. GLA wild-type and mutant plasmids were transfected into mammalian cells; RNA and proteins were extracted for expression and analysis of enzymatic activity. RESULTS: The patient presents the variant p.Asn34Asp in the GLA and had several manifestations of FD since adolescence. The investigation of the deficiency of α-Gal A was initiated due to stage 4 of chronic kidney disease. All family members carrying the novel mutation presented early symptoms, including index case's mother, who received a renal transplant when she was 35 years old. In silico and in vitro analysis confirmed the pathogenic potential of the mutation p.Asn34Asp showing that the enzyme had only 4% of residual activity due to protein misfolding. The ability of the pharmacological chaperone 1-deoxygalactonojirimycin to recover the mutant was confirmed, producing 37.5% of residual activity. CONCLUSION: In this work, we present a novel missense mutation in GLA that leads to the production of a catalytically competent α-Gal A, which is degraded before its delivery to the lysosome, promoting severe manifestations of FD, with a very similar disease course in affected men and women.

Serum ferritin level remains a reliable marker of bone marrow iron stores evaluated by histomorphometry in hemodialysis patients.

BACKGROUND AND OBJECTIVES: As well as being a marker of body iron stores, serum ferritin (sFerritin) has also been shown to be a marker of inflammation in hemodialysis (HD) patients. The aim of this study was to analyze whether sFerritin is a reliable marker of the iron stores present in bone marrow of HD patients. DESIGN: Histomorphometric analysis of stored transiliac bone biopsies was used to assess iron stores by determining the number of iron-stained cells per square millimeter of bone marrow. RESULTS: In 96 patients, the laboratory parameters were hemoglobin = 11.3 +/- 1.6 g/dl, hematocrit = 34.3 +/- 5%, sFerritin = 609 +/- 305 ng/ml, transferrin saturation = 32.7 +/- 22.5%, and C-reactive protein (CRP) = 0.9 +/- 1.4 mg/dl. sFerritin correlated significantly with CRP, bone marrow iron, and time on HD treatment (P = 0.006, 0.001, and 0.048, respectively). The independent determinants of sFerritin were CRP (beta-coef = 0.26; 95% CI = 24.6 to 132.3) and bone marrow iron (beta-coef = 0.32; 95% CI = 0.54 to 2.09). Bone marrow iron was higher in patients with sFerritin >500 ng/ml than in those with sFerritin < or =500 ng/ml. In the group of patients with sFerritin < or =500 ng/ml, the independent determinant of sFerritin was bone marrow iron (beta-coef = 0.48, 95% CI = 0.48 to 1.78), but in the group of patients with sFerritin >500 ng/ml, no independent determinant of sFerritin was found. CONCLUSIONS: sFerritin adequately reflects iron stores in bone marrow of HD patients.

Up-regulation of cyclooxygenase-2 in different grades of acute human renal allograft rejection.

BACKGROUND: Cyclooxygenase-2 (COX-2) is up-regulated by a variety of stimuli that are associated with tissue injury and inflammation. METHODS: The purpose of this study was to analyze COX-2 detection during different grades of acute human renal allograft rejection. COX-2 expressions were analyzed by immunohistochemistry in 74 samples obtained from biopsies with acute rejection of different grades (n= 48), tubular changes (n=13) and from kidney allografts with stable function (n=13). RESULTS: In interstitial area, there was a significant correlation of COX-2 induction in acute rejection in comparison to tubular changes (1.67 vs. 0.76, p=0.02) and stable function (vs. 0.07, p<0.001), as well as in vessels in the group with acute rejection in relation to stable function (1.1 vs. 0, p=0.04). When the group with acute rejection was analyzed in subgroups, there was a clear increase of COX-2 expression from acute rejection grade IB to III in vessels, in inflammatory infiltrating cells in interstitial area and in glomeruli, while borderline and IA grades were intermediate. CONCLUSION: COX-2 is up-regulated during acute human renal allograft rejection according to the severity of acute rejection and could be used as a marker of inflammation in kidney transplantation.

Up-regulation of cyclooxygenase-2 during acute human renal allograft rejection.

BACKGROUND: Cyclooxygenases-1 and -2 (COX-1 and COX-2) are important in renal physiology and in many abnormal states. However, there is poor information about them during renal allograft rejection. The purpose of this study was to analyze cyclooxygenases expression in renal tissue allograft during acute rejection. METHODS: COX-1 and COX-2 transcripts and proteins were analyzed by semi-quantitative RT-PCR and immunohistochemistry in samples from human renal allografts obtained from nephrectomy because of irreversible acute rejection. RESULTS: In samples with acute rejection, we detected higher expression of COX-2 mRNA in comparison with COX-1 (p < 0.001) being COX-2 expression not different from COX-1 in samples from renal allografts without acute rejection. COX-1 and COX-2 localization was in accordance with data described in literature, however COX-2 protein was higher in interstitial cells in the group with rejection than in the group without rejection (p = 0.04). In addition, in samples with acute rejection COX-2 immunoreactivity was more prominent in podocytes (p < 0.001), in proximal tubules (p < 0.001), in collecting duct cells (p = 0.003) and in interstitial cells (p < 0.001) when compared with COX-1. CONCLUSIONS: Our data show that there is an increased production of COX-2 during acute renal rejection.

Apoptosis in kidney and pancreas allograft biopsies.

BACKGROUND: Apoptosis is a particular form of cell death involved in the elimination of somatic cells. In this study, the occurrence of apoptotic cells in kidney and pancreas allograft biopsies was analyzed and correlated with the number of infiltrating macrophages and lymphocytes and granzyme B expression. METHODS: Kidney and pancreas biopsies from patients submitted to simultaneous pancreas-kidney transplantation were classified into three groups: acute rejection, chronic rejection, and transplant cases without evidence of rejection. Formalin-fixed paraffin biopsies were used to identify apoptosis by the terminal deoxynucleotidyl transferase [TdT]-mediated dUTP nick end labeling (TUNEL) method. RESULTS: In normal kidney, only few apoptotic cells were observed. In contrast, in kidney-allograft biopsies, the TUNEL signal was detected in the nuclei of tubular epithelial cells and also in mononuclear cells scattered in the interstitium. In pancreas biopsies, numerous apoptotic cells were detected in acinar cells, in ducts, and occasionally in islets. The number of apoptotic cells in acute pancreas rejection was significantly higher compared with acute rejection of kidney grafts (50+/-14 vs. 21+/-4 cells/mm2; P<0.05). In kidney biopsies, there was a positive correlation between apoptosis and macrophages (r=0.51; P<0.005), and apoptosis versus T lymphocytes (r=0.45; P<0.05). In pancreas biopsies, the number of apoptotic cells correlated only with the number of macrophages (r=0.41; P<0.05). CONCLUSIONS: Apoptosis occurs in kidney and pancreas allograft biopsies, markedly in acute rejection in pancreas biopsies. Although apoptosis may reflect a mechanism of down-regulation of the allograft immune response by eliminating infiltrating cells, the elimination of graft cells may result in graft damage, particularly in pancreas transplantation.

Axoval neuropathy as initial manifestation of primary amyloidosis: report of a case submitted to bone marrow transplantation

A amiloidose é uma síndrome caracterizada pela deposição no meio extracelular de material protéico altamente insolúvel e que pode afetar vários órgãos. Pode ocorrer como doença generalizada e pode ser idiopática (amiloidose primária). Descrevemos o caso de mulher de 48 anos com neuropatia axonal associada a proteinúria na qual a investigação final resultou no diagnóstico de amiloidose primária (AL). Foi submetida a transplante autólogo de medula óssea sem complicações. Discutiremos aspectos relacionados ao diagnóstico da neuropatia e do tratamento atual da AL.

Axoval neuropathy as initial manifestation of primary amyloidosis: report of a case submitted to bone marrow transplantation.

Amyloidosis is a syndrome characterized by deposition of a highly insoluble protein material in the extracellular space that may affect several organs. It may be generalized and idiopathic (primary amyloidosis). We describe the case of a 48 years-old woman with axonal neuropathy associated with proteinuria, whose final investigation resulted in diagnosis of primary amyloidosis (AL). She was submitted to autologous bone marrow transplantation. We discuss some aspects related to diagnosis of neuropathy and current treatment of AL.

Anemia crônica e glomerulopatia secundárias à Doença de Depósito das Cadeias Leves / Chronic anemia and glomerulopathy secondary to Light-chain Deposition Disease

Os autores relatam o caso de uma paciente do sexo feminino, 65 anos de idade, internada com anemia de longa evolução que se associou posteriormente a uma glomerulopatia manifestada por proteinúria, cilindrúria e perda de função renal. As cadeias leves no plasma e na urina estavam elevadas, sobretudo a fração kappa e uma biópsia renal estudada por imunofluorescência e microscopia eletrônica confirmou o diagnóstico de Doença de Depósito das Cadeias Leves. A nefropatia de cadeia leve ocorre pela superprodução de cadeia leve de imunoglobulina produzida por linfócitos B com deposição nas membranas tubulares e no glomérulo.

The autorpresent a case of a 65 year-old female patient, with chronic anemia associated with glomerulopathy manifested as proteinuria, cylindruria and renal failure. There were high serumand urinary levels of light chains and the diagnosis wasperformed by renal biopsy, examined using immunofluorescenceand by electron microscopy that showed light chain paraproteins.Nephropathy of light-chain deposition disease occurs due to anover-production of light chains from immunoglobulins producedby B lymphocytes with a deposit in tubular and glomerularmembranes.

Urinary retinol-binding protein as a prognostic marker in glomerulopathies.

Tubulointerstitial involvement seems to have a decisive influence on the progression of glomerular diseases. We have prospectively evaluated the levels of urinary retinol-binding protein (urRBP), a marker of proximal tubular dysfunction, in patients with different glomerulopathies (GPs) and correlated these levels with disease progression. By studying 238 patients with GPs, we found that urRBP tend to be lower in minimal change disease, glomerular hematuria and poststreptococcal glomerulonephritis as compared to focal segmental glomerulosclerosis, membranous nephropathy and membranoproliferative glomerulonephritis. By following 149 patients for up to 10 years, we have concluded that high levels of urRBP can identify patients who will progress with loss of renal function (defined as doubling of serum creatinine level) and that a urRBP level >1 mg/l was an efficient and independent indicator of poor prognosis as shown by multivariate analysis. This prediction was possible at a time when serum creatinine and creatinine clearance were still in the normal range. Our data suggest that this laboratory test adds important clinical information to the follow-up of GPs.