William L. Clapp

Embryonic Stem Cells Proliferate and Differentiate when Seeded into Kidney Scaffolds

The scarcity of transplant allografts for diseased organs has prompted efforts at tissue regeneration using seeded scaffolds, an approach hampered by the enormity of cell types and complex architectures. Our goal was to decellularize intact organs in a manner that retained the matrix signal for differentiating pluripotent cells. We decellularized intact rat kidneys in a manner that preserved the intricate architecture and seeded them with pluripotent murine embryonic stem cells antegrade through the artery or retrograde through the ureter. Primitive precursor cells populated and proliferated within the glomerular, vascular, and tubular structures. Cells lost their embryonic appearance and expressed immunohistochemical markers for differentiation. Cells not in contact with the basement membrane matrix became apoptotic, thereby forming lumens. These observations suggest that the extracellular matrix can direct regeneration of the kidney, and studies using seeded scaffolds may help define differentiation pathways.

Mouse stem cells seeded into decellularized rat kidney scaffolds endothelialize and remodel basement membranes.

Introduction To address transplant organ shortage, a promising strategy is to decellularize kidneys in a manner that the scaffold retains signals for seeded pluripotent precursor cells to differentiate and recapitulate native structures: matrix-to-cell signaling followed by cell-cell and cell-matrix interactions, thereby remodeling and replacing the original matrix. This would reduce scaffold antigenicity and enable xeno-allografts. Results DAPI-labeled cells in arterial vessels and glomeruli were positive for both endothelial lineage markers, BsLB4 and VEGFR2. Rat scaffold’s basement membrane demonstrated immunolabeling with anti-mouse laminin β1. Labeling intensified over time with 14 day incubations. Conclusion We provide new evidence for matrix-to-cell signaling in acellular whole organ scaffolds that induces differentiation of pluripotent precursor cells to endothelial lineage. Production of mouse basement membrane supports remodeling of host (rat)-derived scaffolds and thereby warrants further investigation as a promising approach for xenotransplantation. Methods We previously showed that murine embryonic stem cells arterially seeded into acellular rat whole kidney scaffolds multiply and demonstrate morphologic, immunohistochemical and gene expression evidence for differentiation. Vascular cell endothelialization was now further tested by endothelial specific BsLB4 lectin and anti-VEGFR2 (Flk1) antibodies. Remodeling of the matrix basement membranes from rat to mouse (“murinization”) was assessed by a monoclonal antibody specific for mouse laminin β1 chain.

Expression of the Ammonia Transporter, Rh C Glycoprotein, in Normal and Neoplastic Human Kidney

Recent studies have identified the presence of a novel Mep/Amt/Rh glycoprotein family of proteins that may play an important role in transmembrane ammonia transport. One of the mammalian members of this family, Rh C glycoprotein (RhCG), transports ammonia, is expressed in distal nephron sites that are critically important for ammonia secretion, exhibits increased expression in response to chronic metabolic acidosis, and originally was cloned as a tumor-related protein. The purpose of our studies was to determine the localization of RhCG in the normal and neoplastic human kidney. Immunoblot analysis of human renal cortical protein lysates demonstrated RhCG protein expression with a molecular weight of approximately 52 kD. Immunohistochemistry revealed both apical and basolateral Rhcg expression in the distal convoluted tubule, connecting segment, and initial collecting tubule and throughout the collecting duct. Co-localization with calbindin-D28k, H(+)-ATPase, aquaporin-2, and pendrin showed that distal convoluted tubule and connecting segment cells, A-type intercalated cells, and non-A, non-B cells express RhCG and that B-type intercalated cells, principal cells, and inner medullary collecting duct cells do not. In renal neoplasms, RhCG was expressed by chromophobe renal cell carcinoma and renal oncocytoma but not by clear cell renal cell carcinoma or by papillary renal cell carcinomas. These studies suggest that RhCG contributes to both apical and basolateral membrane ammonia transport in the human kidney. Furthermore, renal chromophobe renal cell carcinoma and renal oncocytoma seem to originate from the A-type intercalated cell.

Proteinuria: Potential Causes and Approach to Evaluation

Proteinuria may be associated with a renal or systemic disease, or it may be isolated. The latter occurs in asymptomatic patients without evidence of any disease or abnormality of the urine sediment. Isolated proteinuria may be subdivided into two broad groups: (1) benign forms, with a favorable-to-excellent prognosis and (2) persistent forms, some of which have a worrisome prognosis. Functional proteinuria may occur in disorders with altered renal hemodynamics, usually resolves, and is not associated with progressive renal disease. Idiopathic transient proteinuria is typically discovered on routine screening and usually disappears on subsequent testing. In idiopathic intermittent proteinuria, a significant number (50%) of urine samples exhibit abnormal rates of protein excretion. Although structural abnormalities may be observed on renal biopsy, progressive renal insufficiency is unusual. In orthostatic proteinuria, the rate of protein excretion completely normalizes in the recumbent position. Long-term studies show this to be a benign condition. In persistent isolated proteinuria, at least 80% of random urine samples exhibit abnormal protein excretion. This represents a heterogeneous group, but a significant proportion of these patients have prominent renal pathologic findings and progress to serious renal disease. Proteinuria with significant renal disease may be non-nephrotic or nephrotic range. The former does not exclude glomerular disease, but tubulointerstitial or vascular disorders are also likely when proteinuria is less than 2 g/24 hours. Patients with nephrotic-range proteinuria generally have a glomerular disorder. Distinction between benign and more ominous forms of proteinuria requires careful evaluation.

Thromboxane synthase expression in renal transplant patients with rejection

Thromboxane synthase (TS) catalyzes the formation of thromboxane (TxA2) in monocytes/macrophages, platelets, and various tissues. TxA2 is likely to play a role in graft dysfunction due to its vasoconstrictive and platelet aggregatory properties. We studied the expression of TS in 7 normal native kidneys, 29 consecutive renal allograft biopsies (performed for rising serum creatinine, n = 23, and delayed graft function, n = 6), and one transplant nephrectomy specimen with severe acute rejection. TS expression was determined by immunocytochemistry using a monoclonal antibody against human TS, Kon-7. Histologic grading of the transplant biopsy specimens was based on the Banff classification. The degree of TS staining was graded in the glomeruli, interstitium, tubules and vessels from 0 to 3+. Of 29 biopsies, 13 had chronic nephropathy (CN), 6 had acute rejection (AR) with chronic nephropathy (AR/CN), 4 had acute rejection (AR), and 6 had acute tubular necrosis (ATN). TS staining of native kidneys showed sporadic interstitial cells. The biopsy and transplant nephrectomy specimens showed significant staining, predominantly in the glomeruli and interstitium. Positively staining cells appeared to be of macrophage/monocyte lineage by morphology. The mean glomerular staining grade was significantly increased in specimens with AR (2.3 +/- 0.9) and the mean interstitial staining was increased in specimens with AR/CN (2.2 +/- 0.9). Follow-up renal function 6 months post-biopsy showed that patients with higher TS staining grades had a faster decline in graft function. In conclusion, TS expression is increased in patients with acute rejection with or without chronic nephropathy and is associated with more rapid deterioration in function.

Hematology of healthy Florida manatees (Trichechus manatus)

BACKGROUND Hematologic analysis is an important tool in evaluating the general health status of free-ranging manatees and in the diagnosis and monitoring of rehabilitating animals. OBJECTIVES The purpose of this study was to evaluate diagnostically important hematologic analytes in healthy manatees (Trichechus manatus) and to assess variations with respect to location (free ranging vs captive), age class (small calves, large calves, subadults, and adults), and gender. METHODS Blood was collected from 55 free-ranging and 63 captive healthy manatees. Most analytes were measured using a CELL-DYN 3500R; automated reticulocytes were measured with an ADVIA 120. Standard manual methods were used for differential leukocyte counts, reticulocyte and Heinz body counts, and plasma protein and fibrinogen concentrations. RESULTS Rouleaux, slight polychromasia, stomatocytosis, and low numbers of schistocytes and nucleated RBCs (NRBCs) were seen often in stained blood films. Manual reticulocyte counts were higher than automated reticulocyte counts. Heinz bodies were present in erythrocytes of most manatees. Compared with free-ranging manatees, captive animals had slightly lower MCV, MCH, and eosinophil counts and slightly higher heterophil and NRBC counts, and fibrinogen concentration. Total leukocyte, heterophil, and monocyte counts tended to be lower in adults than in younger animals. Small calves tended to have higher reticulocyte counts and NRBC counts than older animals. CONCLUSIONS Hematologic findings were generally similar between captive and free-ranging manatees. Higher manual reticulocyte counts suggest the ADVIA detects only reticulocytes containing large amounts of RNA. Higher reticulocyte and NRBC counts in young calves probably reflect an increased rate of erythropoiesis compared with older animals.

Targeted delivery of let-7a microRNA encapsulated ephrin-A1 conjugated liposomal nanoparticles inhibit tumor growth in lung cancer

MicroRNAs (miRs) are small noncoding RNA sequences that negatively regulate the expression of target genes by posttranscriptional repression. miRs are dysregulated in various diseases, including cancer. let-7a miR, an antioncogenic miR, is downregulated in lung cancers. Our earlier studies demonstrated that let-7a miR inhibits tumor growth in malignant pleural mesothelioma (MPM) and could be a potential therapeutic against lung cancer. EphA2 (ephrin type-A receptor 2) tyrosine kinase is overexpressed in most cancer cells, including MPM and non-small-cell lung cancer (NSCLC) cells. Ephrin-A1, a specific ligand of the EphA2 receptor, inhibits cell proliferation and migration. In this study, to enhance the delivery of miR, the miRs were encapsulated in the DOTAP (N-[1-(2.3-dioleoyloxy)propyl]-N,N,N-trimethyl ammonium)/Cholesterol/DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[cyanur(polyethylene glycol)-2000])-PEG (polyethylene glycol)-cyanur liposomal nanoparticles (LNP) and ephrin-A1 was conjugated on the surface of LNP to target receptor EphA2 on lung cancer cells. The LNP with an average diameter of 100 nm showed high stability, low cytotoxicity, and high loading efficiency of precursor let-7a miR and ephrin-A1. The ephrin-A1 conjugated LNP (ephrin-A1–LNP) and let-7a miR encapsulated LNP (miR–LNP) showed improved transfection efficiency against MPM and NSCLC. The effectiveness of targeted delivery of let-7a miR encapsulated ephrin-A1 conjugated LNP (miR–ephrin-A1–LNP) was determined on MPM and NSCLC tumor growth in vitro. miR–ephrin-A1–LNP significantly increased the delivery of let-7a miR in lung cancer cells when compared with free let-7a miR. In addition, the expression of target gene Ras was significantly repressed following miR–ephrin-A1–LNP treatment. Furthermore, the miR–ephrin-A1–LNP complex significantly inhibited MPM and NSCLC proliferation, migration, and tumor growth. Our results demonstrate that the engineered miR–ephrin-A1–LNP complex is an effective carrier for the targeted delivery of small RNA molecules to lung cancer cells. This could be a potential therapeutic approach against tumors overexpressing the EphA2 receptor.

Primary renal carcinoid tumors: clinicopathologic features of 9 cases with emphasis on novel immunohistochemical findings

Primary renal carcinoid tumors are rare neoplasms. Because of the rarity of these neoplasms, clinicopathologic and immunohistochemical characteristics have not been fully characterized. Immunohistochemistry for renal cell lineage transcription factors, such as paired box gene 2 and paired box gene 8, has not been studied in renal carcinoid tumors and may be useful in demonstrating nephrogenic differentiation. We studied the clinical, morphological, and immunohistochemical features in 9 primary renal carcinoid tumors from multiple institutions with particular emphasis on immunohistochemical findings, in particular, expression of paired box gene 2 and paired box gene 8. All 9 cases expressed at least 1 neuroendocrine marker (CD56, synaptophysin, chromogranin). The renal-associated (paired box gene 2/paired box gene 8), gastrointestinal (caudal-related homeobox-2), and pulmonary/thyroid (thyroid transcription factor-1) transcription factors were not expressed in renal carcinoids (0/9). Of interest, CD99 was expressed in 8 of 9 cases, with the one negative case representing an atypical carcinoid. Perinephric extension and nodal and distant metastases are common. The absence of expression of paired box gene 2 and paired box gene 8, although not conclusive, supports the theory that these are derived from nonnephrogenic elements. CD99 was expressed in almost all cases (8/9); recognition of this could prevent misdiagnosis of a renal primitive neuroectodermal tumor.

Isolation of Tacaribe Virus, a Caribbean Arenavirus, from Host-Seeking Amblyomma americanum Ticks in Florida

Arenaviridae are a family of single stranded RNA viruses of mammals and boid snakes. Twenty-nine distinct mammalian arenaviruses have been identified, many of which cause severe hemorrhagic disease in humans, particularly in parts of sub-Saharan Africa, and in Central and South America. Humans typically become infected with an arenavirus through contact with excreta from infected rodents. Tacaribe virus (TCRV) is an arenavirus that was first isolated from bats and mosquitoes during a rabies surveillance survey conducted in Trinidad from 1956 to 1958. Tacaribe virus is unusual because it has never been associated with a rodent host and since that one time isolation, the virus has not been isolated from any vertebrate or invertebrate hosts. We report the re-isolation of the virus from a pool of 100 host-seeking Amblyomma americanum (lone star ticks) collected in a Florida state park in 2012. TCRV was isolated in two cell lines and its complete genome was sequenced. The tick-derived isolate is nearly identical to the only remaining isolate from Trinidad (TRVL-11573), with 99.6% nucleotide identity across the genome. A quantitative RT-PCR assay was developed to test for viral RNA in host-seeking ticks collected from 3 Florida state parks. Virus RNA was detected in 56/500 (11.2%) of surveyed ticks. As this virus was isolated from ticks that parasitize humans, the ability of the tick to transmit the virus to people should be evaluated. Furthermore, reservoir hosts for the virus need to be identified in order to develop risk assessment models of human infection.

Expression of the ammonia transporter family member, Rh B Glycoprotein, in the human kidney

The ammonia transporter family member, Rh B Glycoprotein (RhBG/Rhbg), is essential for ammonia transport by the rodent kidney, but in the human kidney mRNA but not protein expression has been reported. Because ammonia transport is fundamental for acid-base homeostasis, the current study addressed RhBG expression in the human kidney. Two distinct RhBG mRNA sequences have been reported, with different numbers of consecutive cytosines at nt1265 and thus encoding different carboxy-tails. Sequencing the region of difference in both human kidney and liver mRNA showed eight sequential cytosines, not seven as in some reports. Knowing the correct mRNA sequence for RhBG, we then assessed RhBG protein expression using antibodies against the correct amino acid sequence. Immunoblot analysis demonstrated RhBG protein expression in human kidney and immunohistochemistry identified basolateral RhBG in connecting segment (CNT) and the cortical and outer medullary collecting ducts. Colocalization of RhBG with multiple cell-specific markers demonstrated that that CNT cells and collecting duct type A intercalated cells express high levels of RhBG, and type B intercalated cells and principal cells do not express detectable RhBG. Thus, these studies identify the correct mRNA and thus protein sequence for human RhBG and show that the human kidney expresses basolateral RhBG protein in CNT, type A intercalated cells, and non-A, non-B cells. We conclude that RhBG can mediate an important role in human renal ammonia transport.