Localization of BRCA1 protein in breast cancer tissue and cell lines with mutations.

BACKGROUND: The breast and ovarian cancer susceptibility gene (BRCA1) encodes a tumor suppressor. The BRCA1 protein is found primarily in cell nuclei and plays an important role in the DNA damage response and transcriptional regulation. Deficiencies in DNA repair capabilities have been associated with higher histopathological grade and worse prognosis in breast cancer. METHODS: In order to investigate the subcellular distribution of BRCA1 in tumor tissue we randomly selected 22 breast carcinomas and tested BRCA1 protein localization in frozen and contiguous formalin-fixed, paraffin embedded (FFPE) tissue, using pressure cooker antigen-retrieval and the MS110 antibody staining. To assess the impact of BRCA1 germline mutations on protein localization, we retrospectively tested 16 of the tumor specimens to determine whether they contained the common Ashkenazi Jewish founder mutations in BRCA1 (185delAG, 5382insC), and BRCA2 (6174delT). We also compared co-localization of BRCA1 and nucleolin in MCF7 cells (wild type) and a mutant BRCA1 cell line, HCC1937 (5382insC). RESULTS: In FFPE tissue, with MS110 antibody staining, we frequently found reduced BRCA1 nuclear staining in breast tumor tissue compared to normal tissue, and less BRCA1 staining with higher histological grade in the tumors. However, in the frozen sections, BRCA1 antibody staining showed punctate, intra-nuclear granules in varying numbers of tumor, lactating, and normal cells. Two mutation carriers were identified and were confirmed by gene sequencing. We have also compared co-localization of BRCA1 and nucleolin in MCF7 cells (wild type) and a mutant BRCA1 cell line, HCC1937 (5382insC) and found altered sub-nuclear and nucleolar localization patterns consistent with a functional impact of the mutation on protein localization. CONCLUSIONS: The data presented here support a role for BRCA1 in the pathogenesis of sporadic and inherited breast cancers. The use of well-characterized reagents may lead to further insights into the function of BRCA1 and possibly the further development of targeted therapeutics.

Identifying risk factors in renal allografts before transplant: machine-measured renal resistance and posttransplant allograft survival.

Enhancement of renal allograft function and survival in an era where expanded criteria donors are increasingly used requires validated selection criteria. The goal of this retrospective study was to evaluate the significance of pretransplant donor and allograft parameters to identify risk factors that can be used in a model to predict 1-year allograft outcomes. Donor demographic factors, donor type, and allograft parameters such as biopsy results and machine-measured renal resistance were correlated with 1-year graft outcome. The Kaplan-Meier method was used to estimate graft survival using the categorical predictors of donor type, donor age, and machine measured renal resistance at 1.5, 3, and 5 hours. The log-rank test was used to test the difference in survival curves between cohorts. The Cox regression analysis was used to estimate hazard ratios for machine-measured renal resistance, donor age, donor terminal creatinine level, donor's estimated glomerular filtration rate, cold ischemia time, and percent glomerulosclerosis. The data show that machine-measured renal resistance at 3 and 5 hours has a statistically significant inverse relationship to 1-year graft survival. All other risk factors had no correlation with 1-year graft survival. The machine-measured renal resistance at 3 hours is the earliest significant predictor of 1-year allograft outcome.

Morphologic features of extrahepatic manifestations of hepatitis C virus infection.

Cirrhosis and hepatocellular carcinoma are the prototypic complications of chronic hepatitis C virus infection in the liver. However, hepatitis C virus also affects a variety of other organs that may lead to significant morbidity and mortality. Extrahepatic manifestations of hepatitis C infection include a multitude of disease processes affecting the small vessels, skin, kidneys, salivary gland, eyes, thyroid, and immunologic system. The majority of these conditions are thought to be immune mediated. The most documented of these entities is mixed cryoglobulinemia. Morphologically, immune complex depositions can be identified in small vessels and glomerular capillary walls, leading to leukoclastic vasculitis in the skin and membranoproliferative glomerulonephritis in the kidney. Other HCV-associated entities include porphyria cutanea tarda, lichen planus, necrolytic acral erythema, membranous glomerulonephritis, diabetic nephropathy, B-cell non-Hodgkin lymphomas, insulin resistance, sialadenitis, sicca syndrome, and autoimmune thyroiditis. This paper highlights the histomorphologic features of these processes, which are typically characterized by chronic inflammation, immune complex deposition, and immunoproliferative disease in the affected organ.

Kruppel-like factor 15 (KLF15) is a key regulator of podocyte differentiation.

Podocyte injury resulting from a loss of differentiation is the hallmark of many glomerular diseases. We previously showed that retinoic acid (RA) induces podocyte differentiation via stimulation of the cAMP pathway. However, many podocyte maturity markers lack binding sites for RA-response element or cAMP-response element (CREB) in their promoter regions. We hypothesized that transcription factors induced by RA and downstream of CREB mediate podocyte differentiation. We performed microarray gene expression studies in human podocytes treated with and without RA to identify differentially regulated genes. In comparison with known CREB target genes, we identified Krüppel-like factor 15 (KLF15), a kidney-enriched nuclear transcription factor, that has been previously shown to mediate cell differentiation. We confirmed that RA increased KLF15 expression in both murine and human podocytes. Overexpression of KLF15 stimulated expression of differentiation markers in both wild-type and HIV-1-infected podocytes. Also, KLF15 binding to the promoter regions of nephrin and podocin was increased in RA-treated podocytes. Although KLF15(-/-) mice at base line had minimal phenotype, lipopolysaccharide- or adriamycin-treated KLF15(-/-) mice had a significant increase in proteinuria and podocyte foot process effacement with a reduction in the expression of podocyte differentiation markers as compared with the wild-type treated mice. Finally, KLF15 expression was reduced in glomeruli isolated from HIV transgenic mice as well as in kidney biopsies from patients with HIV-associated nephropathy and idiopathic focal segmental glomerulosclerosis. These results indicate a critical role of KLF15 in mediating podocyte differentiation and in protecting podocytes against injury.

Histopathology of renal failure after heart transplantation: a diverse spectrum.

BACKGROUND: Chronic kidney disease occurs frequently after heart transplantation and is associated with significant morbidity and mortality. Herein we describe the histopathology associated with renal failure in a cohort of heart transplant recipients. METHODS: Over a 4-year period all patients with an estimated GFR <30 ml/min/1.73 m(2) or significant proteinuria were referred to the kidney transplant clinic for clinical evaluation. A percutaneous renal biopsy was performed as part of a standardized evaluation. RESULTS: Eighteen patients underwent renal biopsy 5.8 ± 4.1 years after transplantation. Hypertension (88.9%), Type 2 diabetes (55.6%) and allograft vasculopathy (38.9%) were prevalent. All patients were receiving calcineurin inhibitors. Mean creatinine was 2.9 ± 1.2 mg/dl with an estimated GFR 27.9 ± 9.1 ml/min/1.73 m(2). Eight patients (44%) had proteinuria >1 g per 24 hours. The major histologic findings were nephrosclerosis plus diabetic glomerulopathy (50%), nephrosclerosis and focal segmental glomerulosclerosis (22.2%) and nephrosclerosis alone (22.2%). One patient had direct CNI toxicity consisting of nodular sub-adventitial hyalinosis. Eleven patients (61.1%) had glomerular disease and 11 patients (61.1%) had moderate or severe tubular atrophy. During follow-up, 5 patients (27.8%) started hemodialysis, 4 (22.2%) died, and 2 (11.1%) received a renal transplant. CONCLUSIONS: We observed diverse histologic patterns in this series of renal biopsies suggesting that chronic kidney disease after heart transplantation has a complex and varied pathologic basis. Further defining the renal injuries that precede heart transplantation and predispose to the progression of kidney disease after transplant may assist in treating this population.

Segmentation and thickness measurement of glomerular basement membranes from electron microscopy images.

An algorithm for segmentation and thickness measurement of the glomerular basement membranes (GBM) in electron microscopy kidney images is presented. Differences in intensities and variations between GBM and other components in the image are employed. Regions of extreme intensities such as the black area of blood cells and white areas of urinary spaces are pre-excluded. Areas of sharp edges are either at the GBM borders or unrelated to GBM regions. These non-GBM sharp edges, along with the pre-excluded regions, are used as barriers limiting the size of the fitting circles centered at a location in the image domain to form a two-dimensional function, proportional to the radius of the largest fitting circle, at the location. A local peak in the radius function corresponds to the largest circle in the local area. The set of the combined peaks in two perpendicular directions is calculated before a thinning procedure is applied. After removing the unwanted branches, a centerline of the GBM is produced. The segmentation of the GBM is then straightforward from expanding each point in the centerline to a circle of radius defined by the radius function. The average of the diameters of the circles gives the average GBM thickness. Results of the real GBM images are provided. Visual comparisons from the superimposed GBM boundaries show that the algorithm provides accurate GBM segmentation. The evaluations of the average GBM thicknesses are also compared to those from the manual tracing method.

Paraneoplastic glomerulopathy secondary to retroperitoneal sarcoma: a case report.

Glomerulopathy is a rare form of paraneoplastic disease. We present the second reported case of paraneoplastic glomerulopathy due to a retroperitoneal sarcoma. The patient presented with generalized edema and nephrotic syndrome. CT scan showed two large retroperitoneal masses. One large retroperitoneal mass was resected. Post-operatively, she developed kidney failure and biopsy showed minimal change disease. With steroid therapy, patient's symptoms went into remission. We hypothesize that minimal change paraneoplastic glomerulopathy developed due to damage from cytokines released from a T-cell mediated response to the malignancy.

The variable pathology of kidney disease after liver transplantation.

BACKGROUND: Chronic kidney disease is a frequent complication in orthotopic liver transplant (OLT) recipients, observed in 10% to 60% after 5 years. PATIENTS AND METHODS: We analyzed clinical and pathological data from 81 OLT recipients who developed impaired kidney function with a serum creatinine greater than or equal to 1.5 mg/dL or new proteinuria on dipstick urinalysis. All patients underwent percutaneous kidney biopsy. The most common reason for liver transplantation was hepatitis C virus infection. The mean time until biopsy was 4.89 years. At the time of biopsy, the mean serum creatinine was 2.0 mg/dL, Modified Diet of Renal Disease glomerular filtration rate was 38.7 mL/min, and 24-hr urine protein was 1.37 g. RESULTS: All biopsies demonstrated glomerular abnormalities, 42% showed primary glomerular diseases, and only 16% had evidence of calcineurin inhibitor toxicity. Electron microscopy was performed on 74 biopsies and podocyte effacement was detected in 88%. Mean postbiopsy follow-up was 20 months; eight patients progressed to end-stage renal disease. CONCLUSION: This study demonstrates universal glomerular abnormalities in kidney biopsies after OLT. The pathology is suggestive of diabetic nephropathy and hypertensive change, but there are also specific glomerular disease processes present. There is little calcineurin inhibitor toxicity in this group. These findings underscore the importance of understanding the causes of kidney disease in the constantly changing liver transplant population, and the need to change current management of these patients.

BRCA1 protein and nucleolin colocalize in breast carcinoma tissue and cancer cell lines.

The breast and ovarian cancer susceptibility gene BRCA1 encodes a tumor suppressor. BRCA1 protein, which is involved in DNA damage response, has been thought to be found primarily in cell nuclei. In the present investigation, immunohistological studies of BRCA1 protein in frozen breast cancer tissue and MCF7 and HeLa cell lines revealed BRCA1 expression in both nucleoli and nucleoplasmic foci. Immunoelectron microscopic studies of estrogen-stimulated MCF7 cells demonstrated BRCA1 protein localization in the granular components of the nucleolus. Moreover, immunofluorescence of BRCA1 and nucleolin double-labeling showed colocalization in both nucleoli and nucleoplasmic foci in breast tumor cells and asynchronously growing MCF7 and HeLa cells. Multiparameter analysis of BRCA1 and nucleolin in relation to cell cycle position (DNA content) showed expression during G1-S and persistence of BRCA1 during G2/M. After gamma-irradiation of MCF7 cells, BRCA1 protein dispersed from nucleoli and nucleoplasmic foci to other nucleoplasmic sites, which did not colocalize with nucleolin. Small interfering RNA-mediated knockdown of BRCA1 protein resulted in decreased immunofluorescence staining, which was confirmed by Western blotting. The observed colocalization of BRCA1 and nucleolin raises new possibilities for the nucleoplasm-nucleolus pathways of these proteins and their functional significance.

A semi-automatic algorithm for measurement of basement membrane thickness in kidneys in electron microscopy images.

In this paper, we present a semi-automatic algorithm for measurement of the glomerular basement membrane thickness in electron microscopy kidney images. A string of sparsely spaced points are manually inputted along the central line of the basement membrane (lamina densa) to be measured. The gaps between successive input points are lineally interpolated. A nonlinear mapping is applied to straighten the curved central line. Two distance functions of edges to the central line are constructed. The smooth envelope lines are obtained by repetitive applications of a linear low-pass filtering followed by a comparing and selecting process. The boundaries of the glomerular basement membrane are obtained from the inverse mapping of the envelope functions. The average basement membrane thickness is estimated as the ratio of the basement membrane area to the length of the central line.

Renal gene and protein expression signatures for prediction of kidney disease progression.

Although chronic kidney disease (CKD) is common, only a fraction of CKD patients progress to end-stage renal disease. Molecular predictors to stratify CKD populations according to their risk of progression remain undiscovered. Here we applied transcriptional profiling of kidneys from transforming growth factor-beta1 transgenic (Tg) mice, characterized by heterogeneity of kidney disease progression, to identify 43 genes that discriminate kidneys by severity of glomerular apoptosis before the onset of tubulointerstitial fibrosis in 2-week-old animals. Among the genes examined, 19 showed significant correlation between mRNA expression in uninephrectomized left kidneys at 2 weeks of age and renal disease severity in right kidneys of Tg mice at 4 weeks of age. Gene expression profiles of human orthologs of the 43 genes in kidney biopsies were highly significantly related (R(2) = 0.53; P < 0.001) to the estimated glomerular filtration rates in patients with CKD stages I to V, and discriminated groups of CKD stages I/II and III/IV/V with positive and negative predictive values of 0.8 and 0.83, respectively. Protein expression patterns for selected genes were successfully validated by immunohistochemistry in kidneys of Tg mice and kidney biopsies of patients with IgA nephropathy and CKD stages I to V, respectively. In conclusion, we developed novel mRNA and protein expression signatures that predict progressive renal fibrosis in mice and may be useful molecular predictors of CKD progression in humans.

A case of autoimmune thyroiditis and membranoproliferative glomerulonephritis.

Thyroid hormones play an important role in the growth of the kidney and maintenance of its functions. Prolonged hypothyroidism is known to be accompanied by changes in renal morphology such as thickening of the glomerular and tubular basement membranes as well as increased mesangial matrix. Increased transcapillary leakage of plasma proteins leading to proteinuria and generalized edema is also a known complication of hypothyroidism. In particular, autoimmune thyroiditis is associated with proteinuria. Most previous reports of autoimmune thyroiditis with nephrotic syndrome have demonstrated mixed pathological morphology marked by predominant membranous glomerulopathy. Here we present a patient whose initial presentation with profound hypothyroidism and autoimmune thyroiditis was dominated by nephrotic syndrome secondary to type 1 membranoproliferative glomerulonephritis (MPGN). The association of MPGN and autoimmune thyroiditis is very rare.

Loss of polycystin-1 causes centrosome amplification and genomic instability.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenetic disease predominantly caused by alteration or dysregulation of the PKD1 gene, which encodes polycystin-1 (PC1). The disease is characterized by the progressive expansion of bilateral fluid-filled renal cysts that ultimately lead to renal failure. Individual cysts, even within patients with germline mutations, are genetically heterogeneous, displaying diverse chromosomal abnormalities. To date, the molecular mechanisms responsible for this genetic heterogeneity remain unknown. Using a lentiviral-mediated siRNA expression model of Pkd1 hypomorphism, we show that loss of PC1 function is sufficient to produce centrosome amplification and multipolar spindle formation. These events lead to genomic instability characterized by gross polyploidism and mitotic catastrophe. Following these dramatic early changes, the cell population rapidly converges toward a stable ploidy in which centrosome amplification is significantly decreased, though cytological abnormalities such as micronucleation, chromatin bridges and aneuploidy remain common. In agreement with our in vitro findings, we provide the first in vivo evidence that significant centrosome amplification occurs in kidneys from conditional Pkd1 knockout mice at early and late time during the disease progression as well as in human ADPKD patients. These findings establish a novel function of PC1 in ADPKD pathogenesis and a genetic mechanism that may underlie the intrafamilial variability of ADPKD progression.

[The significance of Goodpasture antigen in hereditary nephritis].

INTRODUCTION: Two types of hereditary nephritis, nonprogressive and progressive, clinically present as asymptomatic haematuria, sometimes combined with proteinuria. At the onset, in both types, light microscopic changes are minimal, immunofluorescence findings are negative, and diagnosis can be made only upon electron microscopic findings that are considered to be specific. OBJECTIVE: The aim of this study was to determine the significance of Goodpasture antigen detection in diagnosis of progressive and nonprogressive hereditary nephritis in its early phase. METHOD: Analysis of renal biopsy specimens was done in patients with hereditary nephritis that were followed from 1990 to 2005. Progression of renal disease was examined in 14 patients with Alport's syndrome, 10 patients with thin basement membrane disease, and 6 patients with unclassified hereditary nephritis diagnosed. For all these cases, indirect immunofluorescence study with serum from a patient with high titer of Goodpasture autoantibodies that recognize the antigenic determinants in human glomerular and tubular basement membrane was performed. RESULTS: In 11 out of 14 cases diagnosed as Alport's syndrome, there was negative staining with Goodpasture serum, and in 3 additional cases with Alport's syndrome, expression of Goodpasture antigen in glomerular basement membrane and thin basement membrane was highly reduced. In all 10 patients with thin basement membrane disease, immunofluorescence showed intensive, bright linear staining with Goodpasture serum along glomerular and tubular basement membrane. In 2 out of 6 patients with unclassified hereditary nephritis, Goodpasture antigen expression was very strong, in one patient it was very reduced, and in 3 patients it was negative. CONCLUSION: The results of our study show that Goodpasture antigen detection plays a very important role in differential diagnosis of progressive and nonpregressive hereditary nephritis, particularly in early phases of the disease.

Transplant glomerulopathy may occur in the absence of donor-specific antibody and C4d staining.

BACKGROUND AND OBJECTIVES: Transplant glomerulopathy (TGP) has been proposed to be a component of chronic antibody-mediated rejection (AMR). We have studied 36 patients with TGP and 51 patients with chronic allograft nephropathy (CAN) but without TGP for C4d staining and donor-specific anti-HLA antibodies (DSA) to investigate the alloantibody-mediated mechanisms. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Allograft biopsies were stained with C4d staining and DSAs were studied by Luminex Flow Beads. Allograft biopsies were done at a mean of 5.3 +/- 5.0 and 5.6 +/- 4.6 yr after transplantation in patients with CAN and TGP, respectively. RESULTS: The mean creatinine level at the time of the biopsy was 2.7 +/- 1.2 mg/dl in each group. Proteinuria of >1.0 g/d was more common in patients with TGP (61 versus 25%; P = 0.002). Whereas three patients with TGP had a history of acute AMR, none of the patients with CAN had. Mean chronicity score of the biopsies were 1.7 +/- 0.7 in patients with CAN and 1.9 +/- 0.8 in patients with TGP. Biopsies from only two (4%) patients with CAN and four (11%) patients with TGP had diffuse C4d positivity. DSA were found in 36% of TGP and 33% of CAN patients. CONCLUSIONS: These results suggest that a substantial number of patients with TGP did not have positive C4d staining or DSA, indicating that a non-alloantibody-mediated process may be involved in the development of TGP in some patients.

Renal transplant patient with polyoma virus bladder infection and subsequent polyoma virus nephropathy.

Polyoma virus nephropathy (PVN) is a significant cause of renal allograft dysfunction in transplant patients. A 58-year-old male received a cadaveric renal transplant and 12 weeks later presented with fever, diarrhea, and dysuria. He was diagnosed with a polyoma virus infection of the bladder by a transurethral bladder biopsy. One year post-transplant, he presented with renal allograft dysfunction and was diagnosed by biopsy with PVN of the non-native kidney. The diagnosis of a polyoma virus infection was confirmed by immunoreactivity to the polyoma T-antigen. We suggest that polyoma virus infection of the bladder be included in the differential diagnosis of urinary dysfunction in post-transplant patients, as such infections might be an under-recognized comorbidity in individuals with PVN.

Expression of p53 in virally infected tubular cells in renal transplant patients with polyomavirus nephropathy.

Polyomavirus (PV) infection is associated with ureteral stenosis, hemorrhagic cystitis, and interstitial nephritis in renal transplant patients. The 3 PVs detected in human beings-BK virus, JC virus, and simian virus 40-each encode highly homologous forms of a large T antigen, a transcriptional and replicational regulatory protein. We describe immunohistochemical findings in 5 renal transplant patients who developed PV nephropathy (PVN) and a sixth patient with both PVN and PV infection of the bladder mucosa. Polyomavirus infection was confirmed by immunohistochemical detection of T antigen in kidney and bladder biopsies. We report on the expression of p53 specific to virally infected cells in all biopsies positive for T antigen. Examination of posttransplant biopsies obtained from these 6 patients before they were diagnosed with PVN revealed no expression of T antigen or p53. Accumulation of p53 in PV-infected cells may occur in response to binding of p53 by T antigen, resulting in stabilization of p53. These results provide the first evidence for intracellular actions of PV T antigen in the context of nonneoplastic diseases.

Later-onset Fabry disease: an adult variant presenting with the cramp-fasciculation syndrome.

BACKGROUND: Classic Fabry disease, an X-linked recessive lysosomal storage disease due to the deficient activity of alpha-galactosidase A, typically presents in early childhood with acroparesthesias, angiokeratomas, hypohidrosis, and corneal dystrophy. The neuropathic pain presumably results from glycosphingolipid accumulation in the vascular endothelium and in small-caliber nerve fibers, and is treatable by enzyme replacement therapy. Later-onset variants with residual alpha-galactosidase A activity lack vascular endothelial involvement and classic symptoms, which lead to the development of cardiac and/or renal disease after the fourth decade of life. OBJECTIVE: To expand the later-onset Fabry phenotype to include cramp-fasciculation syndrome without small-fiber neuropathy. METHODS: A 34-year-old man who presented with chronic exercise-induced pain, fasciculations, and cramps of the feet and legs, and his similarly affected mother, were evaluated. Clinical, biochemical, and molecular studies were performed. RESULTS: Clinical evaluation suggested the diagnosis of Fabry disease, which was confirmed by reduced plasma and leukocyte alpha-galactosidase A activities (8.8% and 13.4% of normal, respectively) due to a missense A143T mutation. His mother was heterozygous for the A143T mutation. CONCLUSION: The presentation of cramps and fasciculations without apparent small-fiber neuropathy expands the phenotype of later-onset Fabry disease.

Differential expression of profibrotic and growth factors in chronic allograft nephropathy.

BACKGROUND: Chronic allograft nephropathy (CAN) is a multifactorial process, where both immunological and nonimmunological factors play roles. Microarrays detect thousands of genes simultaneously. METHODS: We have analyzed gene expression profiles of 16 kidney transplant biopsy samples with CAN by high-density oligonucleotide microarrays, comparing to six normal transplant biopsies. Eight CAN biopsies showed nodular arteriolar hyalinization and one was positive for C4d staining. RESULTS: Hierarchical clustering analysis of the 22 biopsies revealed differential gene expression patterns in CAN versus the control biopsies. However, microarray analysis did not reveal differential gene expression patterns in patients with or without arteriolar hyalinization. Fifty percent of the 100 genes with highest hybridization intensities in a C4d positive sample were related to cellular and humoral immune response. Although 212 genes were upregulated a minimum of 1.5-fold, 112 genes were downregulated in CAN samples. There was differential expression of profibrotic and growth factors that while transforming growth factor-beta induced factor, thrombospondin 1, and platelet derived growth factor-C were up-regulated, vascular endothelial growth factor, epidermal growth factor, and fibroblast growth factors 1 and 9 were downregulated. Selected differentially expressed genes were confirmed in microdissected samples by real-time quantitative PCR. Immunopathologic examination of biopsies revealed strong TGF-beta but decreased glomerular VEGF expression in CAN. CONCLUSION: Microarrays might be an important tool to uncover the mechanisms of multifactorial diseases, such as CAN.