Anti-PODXL Monoclonal antibody, clone USB-2-92 [APC]

Podocalyxin (Podxl) has an essential role in the development and function of the kidney glomerular filtration barrier. It is also expressed by vascular endothelia but perinatal lethality of podxl(-/-) mice has precluded understanding of its function in adult vascular endothelial cells (ECs). In this work, we show that conditional knockout mice with deletion of Podxl restricted to the vascular endothelium grow normally but most die spontaneously around three months of age. Histological analysis showed a nonspecific inflammatory infiltrate within the vessel wall frequently associated with degenerative changes, and involving vessels of different caliber in one or more organs. Podxl-deficient lung EC cultures exhibit increased permeability to dextran and macrophage transmigration. After thrombin stimulation, ECs lacking Podxl showed delayed recovery of VE-cadherin cell contacts, persistence of F-actin stress fibers, and sustained phosphorylation of the ERM complex and activation of RhoA, suggesting a failure in endothelial barrier stabilization. The results suggest that Podxl has an essential role in the regulation of endothelial permeability by influencing the mechanisms involved in the restoration of endothelial barrier integrity after injury. (C) 2016 Elsevier GmbH. All rights reserved.

Background: Established therapies for managing kidney dysfunction such as kidney dialysis and transplantation are limited due to the shortage of compatible donated organs and high costs. Stem cell-based therapies are currently under investigation as an alternative treatment option. As amniotic fluid is composed of fetal urine harboring mesenchymal stem cells (AF-MSCs), we hypothesized that third-trimester amniotic fluid could be a novel source of renal progenitor and differentiated cells. Methods: Human third-trimester amniotic fluid cells (AFCs) were isolated and cultured in distinct media. These cells were characterized as renal progenitor cells with respect to cell morphology, cell surface marker expression, transcriptome and differentiation into chondrocytes, osteoblasts and adipocytes. To test for renal function, a comparative albumin endocytosis assay was performed using AF-MSCs and commercially available renal cells derived from kidney biopsies. Comparative transcriptome analyses of first, second and third trimester-derived AF-MSCs were conducted to monitor expression of renal-related genes. Results: Regardless of the media used, AFCs showed expression of pluripotency-associated markers such as SSEA4, TRA-1-60, TRA-1-81 and C-Kit. They also express the mesenchymal marker Vimentin. Immunophenotyping confirmed that third-trimester AFCs are bona fide MSCs. AF-MSCs expressed the master renal progenitor markers SIX2 and CITED1, in addition to typical renal proteins such as PODXL, LHX1, BRN1 and PAX8. Albumin endocytosis assays demonstrated the functionality of AF-MSCs as renal cells. Additionally, upregulated expression of BMP7 and downregulation of WT1, CD133, SIX2 and C-Kit were observed upon activation of WNT signaling by treatment with the GSK-3 inhibitor CHIR99201. Transcriptome analysis and semiquantitative PCR revealed increasing expression levels of renal-specific genes (e.g., SALL1, HNF4B, SIX2) with gestational time. Moreover, AF-MSCs shared more genes with human kidney cells than with native MSCs and gene ontology terms revealed involvement of biological processes associated with kidney morphogenesis. Conclusions: Third-trimester amniotic fluid contains AF-MSCs of renal origin and this novel source of kidney progenitors may have enormous future potentials for disease modeling, renal repair and drug screening.