Kirk W. Foster

Endocytic delivery of lipocalin-siderophore-iron complex rescues the kidney from ischemia-reperfusion injury

Neutrophil gelatinase-associated lipocalin (Ngal), also known as siderocalin, forms a complex with iron-binding siderophores (Ngal:siderophore:Fe). This complex converts renal progenitors into epithelial tubules. In this study, we tested the hypothesis that Ngal:siderophore:Fe protects adult kidney epithelial cells or accelerates their recovery from damage. Using a mouse model of severe renal failure, ischemia-reperfusion injury, we show that a single dose of Ngal (10 microg), introduced during the initial phase of the disease, dramatically protects the kidney and mitigates azotemia. Ngal activity depends on delivery of the protein and its siderophore to the proximal tubule. Iron must also be delivered, since blockade of the siderophore with gallium inhibits the rescue from ischemia. The Ngal:siderophore:Fe complex upregulates heme oxygenase-1, a protective enzyme, preserves proximal tubule N-cadherin, and inhibits cell death. Because mouse urine contains an Ngal-dependent siderophore-like activity, endogenous Ngal might also play a protective role. Indeed, Ngal is highly accumulated in the human kidney cortical tubules and in the blood and urine after nephrotoxic and ischemic injury. We reveal what we believe to be a novel pathway of iron traffic that is activated in human and mouse renal diseases, and it provides a unique method for their treatment.

Renal Thrombotic Microangiopathy in Mice with Combined Deletion of Endocytic Recycling Regulators EHD3 and EHD4

Eps15 Homology Domain-containing 3 (EHD3), a member of the EHD protein family that regulates endocytic recycling, is the first protein reported to be specifically expressed in the glomerular endothelium in the kidney; therefore we generated Ehd3 –/– mice and assessed renal development and pathology. Ehd3 –/– animals showed no overt defects, and exhibited no proteinuria or glomerular pathology. However, as the expression of EHD4, a related family member, was elevated in the glomerular endothelium of Ehd3 –/– mice and suggested functional compensation, we generated and analyzed Ehd3 –/–; Ehd4 –/– mice. These mice were smaller, possessed smaller and paler kidneys, were proteinuric and died between 3–24 weeks of age. Detailed analyses of Ehd3 –/–; Ehd4 –/– kidneys demonstrated thrombotic microangiopathy (TMA)-like glomerular lesions including thickening and duplication of glomerular basement membrane, endothelial swelling and loss of fenestrations. Other changes included segmental podocyte foot process effacement, mesangial interposition, and abnormal podocytic and mesangial marker expression. The glomerular lesions observed were strikingly similar to those seen in human pre-eclampsia and mouse models of reduced VEGF expression. As altered glomerular endothelial VEGFR2 expression and localization and increased apoptosis was observed in the absence of EHD3 and EHD4, we propose that EHD-mediated endocytic traffic of key surface receptors such as VEGFR2 is essential for physiological control of glomerular function. Furthermore, Ehd3 –/–; Ehd4 –/– mice provide a unique model to elucidate mechanisms of glomerular endothelial injury which is observed in a wide variety of human renal and extra-renal diseases.

Medical education in the digital age: Digital whole slide imaging as an e-learning tool.

The visual aids used in teaching and lecturing to students have clearly evolved over the last several decades. Illustrations on paper were replaced by carousels full of photographic slides, which in turn have been supplanted by Powerpoint™ presentations. At an arguably much slower pace, the method of teaching microscopic anatomy to students of the healthcare professions is also changing. For the majority of medical students graduating before 2000, the microscope and accompanying boxes of glass slides were the standard tools for learning both histology and pathology. Studying for exams also included reviewing kodachromes showing static fields-of-view at predetermined magnifications. Both of these methods of learning required the student to be physically present on campus. Pathology residents, when not learning at the microscope with the attending pathologist, must rely on teaching sets of glass slides that cannot leave the department. Group learning must either be done at a multiheaded microscope (which many times has fewer heads than people present) or by use of video technology attached to the microscope. With the advent of digital whole slide imaging (WSI) over the past several years, there is an opportunity to revolutionize the way teaching and learning are done for all students of medicine including doctors, nurses, medical technicians, histology technicians, cytology technicians, and others. Comparison of Digital Whole Slide Images with Glass Slides There are many advantages to using WSI when compared with traditional glass slides [Table 1]. Digital images can be standardized; all students will study the exact same tissue section. Sections on glass slides are inherently variable in quality and content.[1] Although admittedly subtle in some cases, these variabilities can be eliminated with digital imaging. The quality of the image can also be indefinitely maintained compared to glass slides that are vulnerable to fading, breaking, and vanishing.[2] One very helpful aspect of digital images is the use of a thumbnail image. As students are examining the image at higher magnification, they can always refer to the thumbnail for orientation.[3] This obviously is impossible with glass slides. Glass slides cannot be easily annotated with any precision, relying on relatively crude “dotting” for the purposes of highlighting a certain area of the slide. Digital images can have multiple annotations including arrows, circles, text, etc. placed exactly where needed. Portability is another benefit of using digital images. The use of microscopes obligates a student to remain on campus in order to study, review, etc. With WSI loaded onto a web-based server, study can occur wherever and whenever the student wishes.[2] WSI also makes simultaneous viewing of a particular field-of-view possible; a major advantage over glass slides. Viewing the image on a common computer monitor encourages discussion and collaboration between classmates.[1] Finally, storage of WSI becomes a matter of having enough server memory. As server space becomes less and less expensive, the cost and effort of storing and maintaining both the microscopes and glass slide sets will become comparatively more burdensome.[3] Some disadvantages that have become apparent with WSI include the dependence of the image quality on monitor resolution and the challenge of scanning tissue sections that have artifacts such as folds, etc.[3] Judicious choice of an acceptable non-cost prohibitive monitor is essential for an accurate image. As digital scanners become “smarter”, the flaws in a slide will likely become less of an issue. Table 1 Benefits of digital slide imaging Educational Applications of Digital Whole Slide Imaging The educational applications for this technology are growing. Histology and pathology laboratories and small group study sessions are the early opportunities for medical student use either in a formal computer laboratory or via personal laptops. For those students choosing a pathology residency, WSI can be used for didactic lectures and unknown conferences. Independent study can be encouraged and facilitated with the use of organ-system-based teaching sets, of both neoplastic and non-neoplastic diseases. Diagnostic skills can also be assessed with WSI exams. One academic center looked at the reliability of WSI for measuring resident competency. Using a set of 20 questions based upon 20 whole slide images, junior and senior residents were tested. They determined that the correlation between exam score and months of training was high, the correlation with the resident in-service examination (RISE) was not quite as high, and that the exam could reliably discriminate between junior and senior residents.[4] WSI can also be used for teaching residents or fellows in clinical fields. For pathologists in either academic or non-academic medical centers, WSI can be implemented into both teaching and working interdepartmental conferences such as tumor boards and biopsy conferences (renal, liver, transplant, etc.). For the established pathologist, WSI has become a convenient method of acquiring continuing medical education credits offered through national pathology organization websites. Additional uses for WSI including the creation of electronic books will certainly evolve over time.

Hematopoietic Stem Cell Transplant-Associated Thrombotic Microangiopathy

Hematopoietic stem cell transplant-associated thrombotic microangiopathy (TA-TMA) is a fatal, multifactorial disorder, which may present with thrombocytopenia, hemolysis, acute renal failure, mental status changes and involvement of other organs. The pathogenesis of TA-TMA is complex and includes multiple risk factors such as certain conditioning regimens, calcineurin inhibitors (CNIs), graft-versus-host disease (GVHD), human leukocyte antigen mismatch, and opportunistic infections. The end result of these insults is endothelial injury in the kidney and other organs. Recent studies also indicate a role of complement activation in tissue damage. The lack of sensitive and specific diagnostic tests for TA-TMA often results in delayed diagnosis. Biopsy is not always possible for diagnosis because of the risk of complications such as bleeding. Recently, an emerging role of renal-centered screening approach has been demonstrated, which utilize the monitoring of blood pressure, urine protein, serum lactate dehydrogenase and hemogram for early detection. Therapeutic options are limited, and plasma exchange plays a minor role. Withdrawal of offending agent such as CNIs and the use of rituximab can be effective in some patients. However, the current treatment strategy is suboptimal and associated with high mortality rate. Recently, eculizumab has been utilized in a few patients with good outcomes. Patients, who develop TA-TMA, are also at an increased risk of GVHD, infection, renal, cardiovascular, and other complications, which can contribute to high mortality. Better understanding of molecular pathogenesis, improvement in posttransplant management, leading to early diagnosis, and management of TA-TMA are required to improve outcomes of this fatal entity.

Sixty-five thousand shades of gray: importance of color in surgical pathology diagnoses.

Digital whole slide imaging (WSI) is a diagnostic modality that has gained acceptance as a tool for use in some areas of surgical pathology such as remote consultations. Accurate control of color representation of digitally rendered images of histologic sections is considered an important parameter of WSI. Currently, professional societies, physicians, and other stakeholders are in the process of establishing clinical guidelines outlining the use of these devices, which include color integrity and color calibration of scanners and viewing devices. Although color is a component of surgical pathology diagnoses, it was posited that pathologists could accurately diagnose surgical specimens without color. To test this hypothesis, 5 pathologists were presented breast biopsy specimens from 20 patients consisting of 22 separate tissue specimens and WSI of 158 hematoxylin and eosin-stained slides imaged at ×20. No special stains were included. The pathologists reviewed each case using a 16-bit grayscale monitor and rendered a diagnosis for each case. Diagnoses were compared to the original light microscopy diagnoses and scored for concordance. A 92.7% concordance was observed. Discordant diagnoses represented well-known areas of diagnostic disagreement in breast pathology as well as known limitations of WSI. The research demonstrated that surgical pathologists did not rely primarily on color to render accurate diagnoses of breast biopsy cases but rather used architectural features of tissue and cellular morphology to reach a diagnostic conclusion. This research did not suggest that color is an unimportant factor in pathology diagnosis, but its importance may be overstated.

A Randomized 2×2 Factorial Trial, Part 1: Single-Dose Rabbit Antithymocyte Globulin Induction May Improve Renal Transplantation Outcomes

Background We conducted a randomized and unblinded 2×2 sequential-factorial trial, composed of an induction arm (part 1) comparing single-dose (SD) versus divided-dose rabbit antithymocyte globulin (rATG), and a maintenance arm (part 2) comparing tacrolimus minimization versus withdrawal. We report the long-term safety and efficacy of SD-rATG induction in the context of early steroid withdrawal and tacrolimus minimization or withdrawal. Methods Patients (n=180) received 6 mg/kg rATG, SD or four alternate-day doses (1.5 mg/kg/dose), with early steroid withdrawal and tacrolimus or sirolimus maintenance. After 6 months targeted maintenance levels were tacrolimus, 2 to 4 ng/mL and sirolimus, 4 to 6 ng/mL or, if calcineurin inhibitor–withdrawn, sirolimus 8 to 12 ng/mL with mycophenolate mofetil 2 g two times per day. Primary endpoints were renal function (abbreviated modification of diet in renal disease) and chronic graft histopathology (Banff). Secondary endpoints included patient survival, graft survival, biopsy-proven rejection, and infectious or noninfectious complications. Results Follow-up averaged longer than 4 years. Tacrolimus or sirolimus and mycophenolate mofetil exposure was identical between groups. The SD-rATG associated with improved renal function (2-36 months; P<0.001) in deceased donor recipients. The SD-rATG associated with quicker lymphocyte, CD4 T cell, and CD4-CD8 recovery and fewer infections. Cox multivariate hazard modeling showed divided-dose–rATG (P=0.019), deceased donor (P=0.003), serious infection (P=0.0.018), and lower lymphocyte count (P=0.001) associated with increased mortality. Patients with all four covariates showed a 27-fold increased likelihood of death (P=0.00002). Chronic graft histopathology, rejection rates, and death-censored graft survival were not significantly different between groups. Conclusion The SD-rATG induction improves the 3-year renal function in recipients of deceased donor kidneys. This benefit, along with possibly improved patient survival and fewer infections suggest that how rATG is administered may impact its efficacy and safety.

Visual memory effects on intraoperator study design: determining a minimum time gap between case reviews to reduce recall bias.

OBJECTIVES The objective of this research was to determine test intervals between intraoperator case reviews to minimize the impact of recall. METHODS Three pathologists were presented with a group of 120 slides and subsequently challenged with a study set of 120 slides after 2-week and 4-week intervals. The challenge set consisted of 60 slides seen during the initial review and 60 slides previously unseen within the study. Pathologists rendered a diagnosis for each slide and indicated whether they recalled seeing the slide previously (yes/no). RESULTS Two weeks after having been shown 60 cases from a challenge set of 120 cases, the pathologists correctly remembered 26, 22, and 24 cases or 40% overall. After 4 weeks, the pathologists correctly recalled 31% of cases previously seen. CONCLUSIONS Pathologists were capable of recalling from memory cases seen previously at 2 and 4 weeks. Recall rates may be sufficiently high to affect intraobserver study design.

A Randomized 2x2 Factorial Clinical Trial of Renal Transplantation: Steroid-Free Maintenance Immunosuppression with Calcineurin Inhibitor Withdrawal after Six Months Associates with Improved Renal Function and Reduced Chronic Histopathology

Introduction The two most significant impediments to renal allograft survival are rejection and the direct nephrotoxicity of the immunosuppressant drugs required to prevent it. Calcineurin inhibitors (CNI), a mainstay of most immunosuppression regimens, are particularly nephrotoxic. Until less toxic antirejection agents become available, the only option is to optimize our use of those at hand. Aim To determine whether intensive rabbit anti-thymocyte globulin (rATG) induction followed by CNI withdrawal would individually or combined improve graft function and reduce graft chronic histopathology–surrogates for graft and, therefore, patient survival. As previously reported, a single large rATG dose over 24 hours was well-tolerated and associated with better renal function, fewer infections, and improved patient survival. Here we report testing whether complete CNI discontinuation would improve renal function and decrease graft pathology. Methods Between April 20, 2004 and 4-14-2009 we conducted a prospective, randomized, non-blinded renal transplantation trial of two rATG dosing protocols (single dose, 6 mg/kg vs. divided doses, 1.5 mg/kg every other day x 4; target enrollment = 180). Subsequent maintenance immunosuppression consisted of tacrolimus, a CNI, and sirolimus, a mammalian target of rapamycin inhibitor. We report here the outcome of converting patients after six months either to minimized tacrolimus/sirolimus or mycophenolate mofetil/sirolimus. Primary endpoints were graft function and chronic histopathology from protocol kidney biopsies at 12 and 24 months Results CNI withdrawal (on-treatment analysis) associated with better graft function (p <0.001) and lower chronic histopathology composite scores in protocol biopsies at 12 (p = 0.003) and 24 (p = 0.013) months, without affecting patient (p = 0.81) or graft (p = 0.93) survival, or rejection rate (p = 0.17). Conclusion CNI (tacrolimus) withdrawal at six months may provide a strategy for decreased nephrotoxicity and improved long-term function in steroid-free low immunological risk renal transplant patients. Trial Registration ClinicalTrials.gov NCT00556933

Renal allograft pyelonephritis and fungemia due to Candida krusei

A 61-year-old female was admitted for fever and acute renal failure 6 weeks after receiving a kidney transplant. Her past medical history was notable for CKD stage 5 due to chronic idiopathic tubulointerstitial nephritis. This was diagnosed in 1998 when she presented with an elevated creatinine, sterile pyuria and proteinuria, and underwent native kidney biopsy confirming the diagnosis. She was initially treated with corticosteroids and remained on 5 mg daily of prednisone up until the date of her pre-emptive living related kidney transplant. She had a low-risk immunologic profile with a negative T and B cell crossmatch prior to transplant. Per our center protocol, she received basiliximab for induction, and was maintained on tacrolimus, mycophenolate and prednisone for immunosuppression, with trough FK506 levels ranging between 8 and 12 ng/mL. She was discharged on a standard antimicrobial prophylaxis regimen of trimethoprimsulfamethoxazole and valacyclovir and had a nadir serum creatinine of 1.2 mg/dL following her transplant. Her post-operative course was uneventful until her presentation to the hospital, when she complained of fevers, nausea and vomiting. Her physical examination and

Application of whole slide image markup and annotation for pathologist knowledge capture.

Objective: The ability to transfer image markup and annotation data from one scanned image of a slide to a newly acquired image of the same slide within a single vendor platform was investigated. The goal was to study the ability to use image markup and annotation data files as a mechanism to capture and retain pathologist knowledge without retaining the entire whole slide image (WSI) file. Methods: Accepted mathematical principles were investigated as a method to overcome variations in scans of the same glass slide and to accurately associate image markup and annotation data across different WSI of the same glass slide. Trilateration was used to link fixed points within the image and slide to the placement of markups and annotations of the image in a metadata file. Results: Variation in markup and annotation placement between WSI of the same glass slide was reduced from over 80 μ to less than 4 μ in the x-axis and from 17 μ to 6 μ in the y-axis ( P < 0.025). Conclusion: This methodology allows for the creation of a highly reproducible image library of histopathology images and interpretations for educational and research use.