Multicolor Flow Cytometry and Cytokine Analysis Provides Enhanced Information on Kidney Transplant Biopsies.

INTRODUCTION: Current processing of renal biopsy samples provides limited information about immune mechanisms causing kidney injury and disease activity. We used flow cytometry with transplanted kidney biopsy samples to provide more information on the immune status of the kidney. METHODS: To enhance the information available from a biopsy, we developed a technique for reducing a fraction of a renal biopsy sample to single cells for multicolor flow cytometry and quantitation of secreted cytokines present within the biopsy sample. As proof of concept, we used our technique with transplant kidney biopsy samples to provide examples of clinically relevant immune information obtainable with cytometry. RESULTS: A ratio of CD8+ to CD4+ lymphocytes greater than or equal to 1.2 in transplanted allografts is associated with rejection, even before it is apparent by microscopy. Elevated numbers of CD45 leukocytes and higher levels of interleukin (IL)-6, IL-8, and IL-10 indicate more severe injury. Antibody binding to renal microvascular endothelial cells can be measured and corresponds to antibody-mediated forms of allograft rejection. Eculizumab binding to endothelial cells suggests complement activation, which may be independent of bound antibody. We compared intrarenal leukocyte subsets and activation states to those of peripheral blood from the same donor at the time of biopsy and found significant differences; thus the need for new techniques to evaluate immune responses within the kidney. CONCLUSION: Assessment of leukocyte subsets, renal microvascular endothelial properties, and measurement of cytokines within a renal biopsy by flow cytometry enhance understanding of pathogenesis, indicate disease activity, and identify potential targets for therapy.

A Novel Three-Dimensional Human Peritubular Microvascular System.

Human kidney peritubular capillaries are particularly susceptible to injury, resulting in dysregulated angiogenesis, capillary rarefaction and regression, and progressive loss of kidney function. However, little is known about the structure and function of human kidney microvasculature. Here, we isolated, purified, and characterized human kidney peritubular microvascular endothelial cells (HKMECs) and reconstituted a three-dimensional human kidney microvasculature in a flow-directed microphysiologic system. By combining epithelial cell depletion and cell culture in media with high concentrations of vascular endothelial growth factor, we obtained HKMECs of high purity in large quantity. Unlike other endothelial cells, isolated HKMECs depended on high vascular endothelial growth factor concentration for survival and growth and exhibited high tubulogenic but low angiogenic potential. Furthermore, HKMECs had a different transcriptional profile. Under flow, HKMECs formed a thin fenestrated endothelium with a functional permeability barrier. In conclusion, this three-dimensional HKMEC-specific microphysiologic system recapitulates human kidney microvascular structure and function and shows phenotypic characteristics different from those of other microvascular endothelial cells.

CMS-required visits adversely impact Seattle area hemodialysis program.

Chronic hemodialysis in Seattle developed with a highly patient-centric approach, providing patients the option of dialyzing in neighborhood units under the administration of a non-profit organization, the Northwest Kidney Center (NKC). This study examined the effects of a requirement in the 2009 Conditions for Coverage that in-center hemodialysis patients be visited quarterly during their treatment session. A retrospective analysis of quality outcome indicators for patients managed at the University of Washington Medical Center and dialyzing at NKC in 2008 and 2010 found that dialysis adequacy (eKt/V), phosphorus, albumin, and hemoglobin were not improved by quarterly dialysis visits. Instead, dialysis unit visits adversely impacted patient-nephrologist relationships and diverted nephrologists from other patient care activities.

Proliferative potential of human kidney endothelial cells: bone marrow-derived cells may not be required for high proliferation.

BACKGROUND: Proliferative potential of a single cell, defined as the number of progeny it gives rise to, has been used to define a hierarchy of endothelial progenitor cells in blood. Cells with high proliferative potential are presumed to have greater capacity for endothelium repair. Based on results with commercially available endothelial cells, it has been proposed that a proliferative hierarchy of endothelial cells also exists within blood vessels. It is unknown whether such vessel-derived highly proliferative endothelial cells originate from the bone marrow or whether the supply of precursors is limited to pre-existing cells that reside within vessels. METHODS: In this study, we isolated normal human renal microvascular endothelial cells (RMEC) and larger cortical vessel endothelial cells (EC) by flow cytometry based on differential expression of human leucocyte antigen (HLA)-DR, and evaluated the proliferative potential of single cells. To determine if highly proliferative clones might derive from bone marrow recruits, HLA-DR expression on RMEC from transplanted kidneys was evaluated using antibodies that distinguish donor cells from recipient cells. RESULTS: We found the proliferative potential of kidney endothelial cells diverse and variable. Subcloning indicated that proliferative potential was determined by epigenetic events. In transplanted kidneys affected with a variety of different injuries, RMEC were donor derived. CONCLUSIONS: We conclude that endothelial cells of high proliferative potential exist within human renal blood vessels, even in individuals into their eighth decade of life, and that highly proliferative endothelial cells are unlikely to be bone marrow derived.

Reoperative thyroidectomy for benign thyroid disease.

BACKGROUND: Subtotal thyroidectomy for benign thyroid disease (BTD) may lead to delayed recurrence, thus necessitating reoperative surgery. We describe our experience with reoperative thyroidectomy for BTD and recommendations for definitive primary management. METHODS: Patients undergoing thyroid surgery between 2003 and 2007 by a single surgeon were prospectively assessed. Numerous clinical parameters were evaluated, including time interval between primary and reoperative surgery and complications. RESULTS: In all, 321 thyroidectomies were identified: 45 were reoperative and 22 were related to BTD after primary surgery done elsewhere. Median interval between the primary and reoperative procedure was 8.5 years. No recurrences followed total thyroidectomy or total thyroid lobectomy. There were no cases of permanent or transient recurrent laryngeal nerve (RLN) injury related to reoperative surgery. There was 1 case of transient hypocalcemia. CONCLUSIONS: Although reoperative thyroidectomy can be performed safely in the hands of experienced surgeons, a thorough initial surgical procedure should obviate the need for exposure to this additional risk.

Findings from a public thyroid screening protocol: ultrasound and disease characteristics.

OBJECTIVES: A public thyroid screening protocol incorporating ultrasonography was developed and implemented as a feature of the National Thyroid Cancer Awareness month. Findings and lessons learned are described. METHODS: Prospective analysis of participants in a 1-day thyroid screening protocol and review of findings and referrals generated during the screening process. RESULTS: A total of 39 patients participated in the thyroid-screening protocol. Thirty-two (82%) patients were female and 7 (18%) were male, with an overall mean age of 52.9 +/- 14.1 years (range: 20-79). Seventeen (44%) patients indicated a known history of thyroid pathology, and 5 (13%) patients reported a family history of thyroid disease. The most common complaints offered on a patient intake survey were weight gain (38%) and dysphagia (36%). Thirty patients (77%) underwent thyroid ultrasound (US). The majority of patients (69%) had an abnormal US; the most common abnormality found was multinodular goiter (21%). Eighteen participants were referred to endocrinology for further evaluation, 13 have been evaluated and 3 patients have had fine-needle aspirations performed. Two patients have undergone thyroid surgery. The majority of patients (67%) believed that the thyroid-screening increased their awareness and knowledge of thyroid and head and neck cancer. CONCLUSIONS: A public thyroid screening activity proved to be a valuable mechanism for the dual purpose of identifying individuals with thyroid pathology needing further evaluation, and increased public awareness and knowledge of thyroid and head and neck cancer. Additional value related to the provision of a community service and opportunity to increase experience with ultrasonography.

Noninvasive measurement of protein aggregation by mutant huntingtin fragments or alpha-synuclein in the lens.

Many diverse human diseases are associated with protein aggregation in ordered fibrillar structures called amyloid. Amyloid formation may mediate aberrant protein interactions that culminate in neurodegeneration in Alzheimer, Huntington, and Parkinson diseases and in prion encephalopathies. Studies of protein aggregation in the brain are hampered by limitations in imaging techniques and often require invasive methods that can only be performed postmortem. Here we describe transgenic mice in which aggregation-prone proteins that cause Huntington and Parkinson disease are expressed in the ocular lens. Expression of a mutant huntingtin fragment or alpha-synuclein in the lens leads to protein aggregation and cataract formation, which can be monitored in real time by noninvasive, highly sensitive optical techniques. Expression of a mutant huntingtin fragment in mice lacking the major lens chaperone, alphaB-crystallin, markedly accelerated the onset and severity of aggregation, demonstrating that the endogenous chaperone activity of alphaB-crystallin suppresses aggregation in vivo. These novel mouse models will facilitate the characterization of protein aggregation in vivo and are being used in efficient and economical screens for chemical and genetic modifiers of disease-relevant protein aggregation.

Laryngeal nerve monitoring and minimally invasive thyroid surgery: complementary technologies.

OBJECTIVE: To determine the feasibility of the combined use of laryngeal nerve monitoring and minimally invasive thyroid surgery. DESIGN: Prospective, nonrandomized analysis of single-surgeon experience. SETTING: Academic institution. PATIENTS: Consecutive series of patients undergoing both minimally invasive thyroid surgery and laryngeal nerve monitoring. MAIN OUTCOME MEASURES: Incision length and incidence of temporary or permanent laryngeal nerve injury. RESULTS: Two hundred eighty-three patients underwent thyroid surgery at the Medical College of Georgia, Augusta, between January 2004 and November 2006. Some type of minimal-access approach (endoscopic or nonendoscopic) was used in 137 cases (48.4%) in which general anesthesia was administered. Laryngeal nerve monitoring was performed in 73 (53.3%) of these 137 cases, although the proportion of cases in which it was performed increased significantly from 8.7% (2 of 23 cases) in 2004 to 95.2% (58 of 61 cases) in 2006 (P < .001). There were no cases of permanent nerve injury. The incidence of temporary recurrent laryngeal nerve paresis was 4.3% (4 of 92 nerves at risk) in the cases in which laryngeal nerve monitoring was used and 6.0% (5 of 84 nerves at risk) in the cases in which the nerve was visually identified without use of a monitor. This difference failed to reach statistical significance (P = .73), which may reflect an insufficient sample size. CONCLUSION: Monitoring of the laryngeal nerves is feasible in minimal-access thyroid surgery and may serve as a meaningful adjunct to the visual identification of nerves.

Senataxin, the yeast Sen1p orthologue: characterization of a unique protein in which recessive mutations cause ataxia and dominant mutations cause motor neuron disease.

A severe recessive cerebellar ataxia, Ataxia-Oculomotor Apraxia 2 (AOA2) and a juvenile onset form of dominant amyotrophic lateral sclerosis (ALS4) result from mutations of the Senataxin (SETX) gene. To begin characterization this disease protein, we developed a specific antibody to the DNA/RNA helicase domain of SETX. In murine brain, SETX concentrates in several regions, including cerebellum, hippocampus and olfactory bulb with a general neuronal expression profile, colocalizing with NeuN. In cultured cells, we found that SETX was cytoplasmically diffuse, but in the nucleus, SETX was punctate, colocalizing with fibrillarin, a marker of the nucleolus. In differentiated non-cycling cells, nuclear SETX was not restricted to the nucleolus but was diffuse within the nucleoplasm, suggesting cell-cycle-dependent localization. SETX missense mutations cluster within the N-terminus and helicase domains. Flag tagging at the N-terminus caused protein mislocation to the nucleoplasm and failure to export to the cytoplasm, suggesting that the N-terminus may be essential for correct SETX localization. We report here the first characterization of SETX protein, which may provide future insights into a new mechanism leading to neuron death.

SIMPLE interacts with NEDD4 and TSG101: evidence for a role in lysosomal sorting and implications for Charcot-Marie-Tooth disease.

Mutation of the SIMPLE gene (small integral membrane protein of the lysosome/late endosome) is the molecular basis of Charcot-Marie-Tooth disease type 1C (CMT1C), a demyelinating peripheral neuropathy. Although the precise function of SIMPLE is unknown, prior reports suggest it localizes to the lysosome/late endosome. Furthermore, murine Simple interacts with Nedd4 (neural precursor cell expressed, developmentally downregulated 4), an E3 ubiquitin ligase that is important for regulating lysosomal degradation of plasma membrane proteins. To bring insights into the biochemical function of human SIMPLE, we confirmed that human SIMPLE interacts with NEDD4 and also report a novel interaction with tumor susceptibility gene 101 (TSG101), a class E vacuolar sorting protein. TSG101 is known to function downstream of NEDD4, sorting ubiquitinated substrates into multivesicular bodies (MVBs), which then deliver their cargo into the lysosomal lumen for degradation. Given the interaction with NEDD4 and TSG101, and the localization of SIMPLE along the lysosomal degradation pathway, we hypothesize that SIMPLE plays a role in the lysosomal sorting of plasma membrane proteins. We examine three CMT1C-associated SIMPLE mutations and show that they do not affect the interaction with NEDD4 or TSG101, nor do they lead to altered subcellular localization.

Normal human kidney HLA-DR-expressing renal microvascular endothelial cells: characterization, isolation, and regulation of MHC class II expression.

Human, but not murine, renal peritubular and glomerular capillaries constitutively express class II major histocompatibility (MHC) proteins at high levels in normal human kidney. Expression of class II proteins on renal microvascular endothelial cells (RMEC) makes it available to circulating lymphocytes and imparts a surveillance capacity to RMEC for controlling inflammatory responses. In this report, the co-expression of HLA-DR and the endothelial marker CD31 are used to identify RMEC as a distinct population of cells within a standard renal biopsy using flow cytometry. A three-laser, multicolor flow cytometry analysis using Alexa dyes, developed for characterizing the expression of cell surface antigens, identifies RMEC as a population separate from HLA-DR-expressing leukocytes. HLA-DR RMEC co-express HLA-DP and HLA-DQ. RMEC also express the T cell costimulatory factor CD58 but not CD80, CD86, or CD40. On the basis of high HLA-DR expression, RMEC are isolated for culture using fluorescence-activated cell sorting and magnetic beads. Cultured RMEC require normal basal physiologic concentrations of gamma interferon (gammaIFN) to maintain HLA protein expression. This expression is regulated by CIITA, the MHC class II-specific transcription factor. Four tissue-specific promoters have been described for CIITA. In freshly isolated RMEC, RT-PCR and hybridization using specific oligonucleotide probes to CIITA promoter sequences identify only the statin-sensitive gammaIFN-induced promoter IV of CIITA. Therefore, the constitutive expression of HLA-DR on RMEC in normal human kidney is located in a position for immune surveillance, depends on basal physiologic concentrations of gammaIFN, and may be amenable to regulation with statins.