Immunohistochemical changes in kidney glomerular and tubular proteins caused by rattlesnake (Crotalus vegrandis) venom

Renal damage is an important cause of death in patients who have survived the early effects of severe crotalid envenomation. Extracellular matrix of renal tissue is altered by Crotalus toxin activities. The aim of this study was to describe how cytoskeletal proteins and basal membrane components undergo substantial alterations under the action of Crotalus vegrandis crude venom and its hemorrhagic fraction (Uracoina-1) in mice. To detect the proteins in question, the immunoperoxidase method with monoclonal and polyclonal antibodies was used. Cell types within renal lesions were characterized by phenotypic identification, by means of immunohistologic analysis of marker proteins using different primary antibodies against mesangial cells, endothelial cells, cytoskeletal proteins (intermediate filament), extracellular matrix and basal membranes. Samples for morphological study by standard procedures (biotin-streptavidin-peroxidase technique) using light microscopy were processed. Positive and negative controls for each antigen tested in the staining assay were included. After crude venom and hemorrhagic fraction inoculation of mice, the disappearance of cytoskeletal vimentin and desmin and collagen proteins in the kidney was observed. In extracellular matrix and basal membranes, collagen type IV from envenomed animals tends to disappear from 24 h to 120 h after venom injection.

A low-cost method to test cytotoxic effects of Crotalus vegrandis (Serpentes: Viperidae) venom on kidney cell cultures

The pathogenesis of the renal lesion upon envenomation by snakebite has been related to myolysis, hemolysis, hypotension and/or direct venom nephrotoxicity caused by the venom. Both primary and continuous cell culture systems provide an in vitro alternative for quantitative evaluation of the toxicity of snake venoms. Crude Crotalus vegrandis venom was fractionated by molecular exclusion chromatography. The toxicity of C. vegrandis crude venom, hemorrhagic, and neurotoxic fractions were evaluated on mouse primary renal cells and a continuous cell line of Vero cells maintained in vitro. Cells were isolated from murine renal cortex and were grown in 96 well plates with Dulbecco's Modified Essential Medium (DMEM) and challenged with crude and venom fractions. The murine renal cortex cells exhibited epithelial morphology and the majority showed smooth muscle actin determined by immune-staining. The cytotoxicity was evaluated by the tetrazolium colorimetric method. Cell viability was less for crude venom, followed by the hemorrhagic and neurotoxic fractions with a CT50 of 4.93, 18.41 and 50.22 µg/mL, respectively. The Vero cell cultures seemed to be more sensitive with a CT50 of 2.9 and 1.4 µg/mL for crude venom and the hemorrhagic peak, respectively. The results of this study show the potential of using cell culture system to evaluate venom toxicity.

Treatment of Hepatitis C Virus Infection in Kidney Transplant Recipients: Case Report

Chronic hepatitis C virus (HCV) infection exists in a large proportion of patients undergoing renal transplantation. Nowadays it is not considered to be an absolute contraindication to transplantation; however, it is associated with an increased risk for the patient and accounts for a shorter half-life of the renal allograft. We present three transplant recipients who displayed serious hepatic dysfunction after renal transplantation due to an HCV infection. In two of these cases, the liver biopsies established the diagnosis of FCH. In the third case, the liver biopsy was compatible with the early stages of FCH. All patients were started on peg-interferon alfa 2-b and ribavirin with subsequent normalization of hepatic function and early complete viral responses.

Specificity and Sensitivity of Screening for Anti-HLA Antibodies in Kidney Allograft Dysfunction

BACKGROUND: Prospective testing for posttransplant circulating anti-HLA antibodies seems to be a critical noninvasive tool, but confirmatory data are lacking. MATERIALS AND METHODS: Over the last 3 years, peritubular capillary (PTC) C4d deposition was prospectively sought by an immunofluorescence technique applied to frozen tissue in biopsies obtained for allograft dysfunction. Screening for circulating anti-HLA class I/II alloantibodies (AlloAb) by the flow cytometric test was performed simultaneously. RESULTS: We evaluated 132 sets of biopsies and simultaneous serum samples. PTC C4d deposition was demonstrated in 15.9% (21/132) of biopsies. Circulating anti-HLA I/II AlloAb were detected in 25% (33/132) of serum samples. Employing receiver-operator characteristic (ROC) curves for all C4d-positive biopsies, screening for AlloAb showed a global specificity of 82% and sensitivity of 61.9%. When this analysis was restricted to biopsies obtained in the first month posttransplantation, the sensitivity increased to 81.8%, but the specificity decreased to 76.9%. After the first month posttransplantation, we observed sensitivity of 40.0% and a specificity of 86.4%. In the first month posttransplantation, all patients with a diagnosis of acute antibody-mediated rejection displayed circulating anti-HLA class I/II, but not always at the same time as the C4d-positive biopsy. CONCLUSIONS: In the first month posttransplantation, prospective monitoring of anti-HLA antibodies may be useful. The high sensitivity allows the identification of patients at risk, affording an earlier diagnosis of antibody-mediated rejection. After the first month, the test can be used to evaluate allograft dysfunction episodes, since positivity is highly suggestive of an antibody-mediated process.

Leukopenia in Kidney Transplant Patients with the Association of Valganciclovir and Mycophenolate Mofetil

Cytomegalovirus (CMV) is the most common viral infection after transplantation. Valganciclovir (VGC) is established for prophylaxis and treatment of CMV infections, but leukopenia which appears in 10% to 13% (severe in 4.9%) is the principal side effect. We have recently noted an increased incidence of leukopenia and severe neutropenia among our renal transplant patients and thought to identify the associated factors. We conducted a retrospective analysis of all kidney transplantations performed between January 2005 and December 2006. All patients received mycophenolate mofetil (MMF), tacrolimus, and steroids. VGC was used for targeted prophylaxis and preemptive therapy of CMV infection, with doses adjusted to renal function. Of the 64 patients undergoing renal transplantation 13 (20.3%) developed leukopenia within 3 +/- 2 months after transplantation with severe neutropenia in 5 (7.8%). All patients were on MMF and VGC (VGC 605 +/- 296 mg/d). Leukopenia was significantly associated with simultaneous liver-kidney transplantation and with second kidney transplantations (P < .01). The incidence of leukopenia was higher among patients under VGC since day 1 of transplantation (P = .008) with maximal incidence observed among patients prescribed 900 mg/d as opposed to those on lower doses (P < .01). There was no increase in CMV infection among patients with a low dose of VGC. No patient developed clinical CMV disease. In conclusion, VGC prophylaxis was associated with an increased frequency of leukopenia on MMF-tacrolimus treated patients or regimens. Low-dose VGC for CMV prophylaxis appeared to be as effective as high-dose treatment, and associated less frequently with leukopenia and neutropenia.

Prevalence of hepatitis C virus infection in quilombo remnant communities in Central Brazil

In order to determine the prevalence of hepatitis C virus (HCV) infection in quilombo remnant communities in Central Brazil, 1,007 subjects were interviewed in all 12 communities existing in Mato Grosso do Sul State, Central Brazil. Blood samples were collected and sera were tested for anti-HCV by enzyme-linked immunosorbent assay. Positive samples were retested for confirmation using a line immunoassay and were also subjected to HCV RNA detection. The prevalence of HCV infection was 0.2%. This finding shows a low prevalence of HCV infection in quilombo remnant communities in Central Brazil.

Zero-Time Kidney Histology Predicts Early Graft Function

Our purposes are to determine the impact of histological factors observed in zero-time biopsies on early post transplant kidney allograft function. We specifically want to compare the semi-quantitative Banff Classification of zero time biopsies with quantification of % cortical area fibrosis. Sixty three zero-time deceased donor allograft biopsies were retrospectively semiquantitatively scored using Banff classification. By adding the individual chronic parameters a Banff Chronic Sum (BCS) Score was generated. Percentage of cortical area Picro Sirius Red (%PSR) staining was assessed and calculated with a computer program. A negative linear regression between %PSR/ GFR at 3 year post-transplantation was established (Y=62.08 +-4.6412X; p=0.022). A significant negative correlation between arteriolar hyalinosis (rho=-0.375; p=0.005), chronic interstitial (rho=0.296; p=0.02) , chronic tubular ( rho=0.276; p=0.04) , chronic vascular (rho= -0.360;P=0.007), BCS (rho=-0.413; p=0.002) and GFR at 3 years were found. However, no correlation was found between % PSR, Ci, Ct or BCS. In multivariate linear regression the negative predictive factors of 3 years GFR were: BCS in histological model; donor kidney age, recipient age and black race in clinical model. The BCS seems a good and easy to perform tool, available to every pathologist, with significant predictive short-term value. The %PSR predicts short term kidney function in univariate study and involves extra-routine and expensive-time work. We think that %PSR must be regarded as a research instrument.

C4d Presence in Kidney Allograft Biopsy: Sensitivity and Specifity of Immunoperoxidase vs Immunofluorescence

OBJECTIVES: Evaluate the sensitivity/specificity of immunoperoxidase method in comparison with the standard immunofluorescence. MATERIAL AND METHODS: Retrospective review of 87 biopsies made for allograft dysfunction. Immunofluorescence (IF) was performed in frozen allograft biopsies using monoclonal antibody anti-C4d from Quidel®. The indirect immunoperoxidase (IP) technique was performed in paraffin-embebbed tissue with polyclonal antiserum from Serotec®. Biopsies were independently evaluated by two nephropathologist according Banff 2007 classification. RESULTS: By IF, peritubular C4d deposition were detected in 60 biopsies and absent in 27 biopsies. The evaluation of biopsy by IP was less precise due to the presence of background and unspecific staining. We find 13.8% (12/87) of false negative and Banff classification concordance in 79.3% (69/87) of cases (table1). The ROC curve study reveal a specificity of 100% and sensitivity of 80.0 % of IP method in relation to the gold standard (area under curve:0.900; 95% Confidence interval :0.817-0.954; p=0.0001). Banff Classification C4d Cases Immunofluorescence Immunoperoxidase n =87 Diffuse Negative 3 (3.4%) Focal Negative 9 (10.3%) Negative Negative 27 (31.0%) Diffuse Diffuse 33 (37.9%) Focal Focal 9 (10.3%) Diffuse Focal 6 (6.9%) CONCLUSION: The IP method presents a good specificity, but lesser sensitivity to C4d detection in allograft dysfunction. The evaluation is more difficult, requiring more experience of the observer than IF method. If frozen tissue is unavailable, the use of IP for C4d detection is acceptable.

ACUTE KIDNEY INJURY CAUSED BY Crotalus AND Bothrops SNAKE VENOM: A REVIEW OF EPIDEMIOLOGY, CLINICAL MANIFESTATIONS AND TREATMENT

SUMMARY Ophidic accidents are an important public health problem due to their incidence, morbidity and mortality. An increasing number of cases have been registered in Brazil in the last few years. Several studies point to the importance of knowing the clinical complications and adequate approach in these accidents. However, knowledge about the risk factors is not enough and there are an increasing number of deaths due to these accidents in Brazil. In this context, acute kidney injury (AKI) appears as one of the main causes of death and consequences for these victims, which are mainly young males working in rural areas. Snakes of the Bothrops and Crotalus genera are the main responsible for renal involvement in ophidic accidents in South America. The present study is a literature review of AKI caused by Bothrops and Crotalus snake venom regarding diverse characteristics, emphasizing the most appropriate therapeutic approach for these cases. Recent studies have been carried out searching for complementary therapies for the treatment of ophidic accidents, including the use of lipoic acid, simvastatin and allopurinol. Some plants, such as Apocynaceae, Lamiaceae and Rubiaceae seem to have a beneficial role in the treatment of this type of envenomation. Future studies will certainly find new therapeutic measures for ophidic accidents.

Mineral and Bone Disease (MBD) on a Kidney Transplant Patient

A 50-year-old post-menopausal recipient of a kidney allograft with bone pain, osteoporosis, persistent hypercalcaemia and elevated parathormone (PTH) levels, despite a satisfactory graft function, was treated with bisphosphonates and cinacalcet starting, respectively, 5 and 6 months after renal transplantation (RT). Sixteen months after treatment, there was improvement of bone mineral density (BMD) measured by dualenergy X-ray absorptiometry (DEXA). A bone biopsy was taken, unveiling a surprising and worrisome result. Post-RT bone disease is different from classic CKD-MBD and should be managed distinctly, including, in some difficult cases, an invasive evaluation through the performance of a bone biopsy, as suggested in the KDIGO guidelines.

OCULAR SYPHILIS IN A KIDNEY TRANSPLANT RECIPIENT

We present a case of ocular syphilis after a renal transplantation involving progressive vision loss without clinically identifiable ocular disease. Electroretinography showed signs of ischemia, especially in the internal retina. A serological test was positive for syphilis. Lumbar puncture revealed lymphocytic meningitis and a positive serologic test for syphilis in the cerebrospinal fluid. The patient was treated with penicillin, and had a quick vision improvement. In the case of transplant recipients, clinicians should always consider the diagnosis of ocular syphilis in cases with unexplained visual acuity decrement, as this condition may cause serious complications if not treated.