Antibodies to kidney endothelial cells contribute to a "leaky" glomerular barrier in patients with chronic kidney diseases.

Anti-endothelial cell antibodies (AECA) have been reported to cause endothelial dysfunction, but their clinical importance for tissue-specific endothelial cells is not clear. We hypothesized that AECA reactive with human kidney endothelial cells (HKEC) may cause renal endothelial dysfunction in patients with chronic kidney diseases. We report that a higher fraction (56%) of end-stage renal disease (ESRD) patients than healthy controls (5%) have AECA reactive against kidney endothelial cells (P <0.001). The presence of antibodies was associated with female gender (P < 0.001), systolic hypertension (P < 0.01), and elevated TNF-α (P < 0.05). These antibodies markedly decrease expression of both adherens and tight junction proteins VE-cadherin, claudin-1, and zonula occludens-1 and provoked a rapid increase in cytosolic free Ca(2+) and rearrangement of actin filaments in HKEC compared with controls. This was followed by an enhancement in protein flux and phosphorylation of VE-cadherin, events associated with augmented endothelial cell permeability. Additionally, kidney biopsies from ESRD patients with AECA but not controls demonstrated a marked decrease in adherens and tight junctions in glomerular endothelium, confirming our in vitro data. In summary, our data demonstrate a causal link between AECA and their capacity to induce alterations in glomerular vascular permeability.

Isolation and characterization of human primary enterocytes from small intestine using a novel method.

Cell culture studies of enterocytes are important in many fields. However, there are difficulties in obtaining cell lines from adult human intestine, such as microbial contamination of cultures from the tissue samples, short life span of enterocytes, overgrowth of mesenchymal cells, etc. Various model used to obtain adult intestinal cell lines are very complex requiring use of feeder layer or gel matrices. The aim of this study was to establish a novel method for the simple and reproducible isolation of human enterocytes. Enterocytes were isolated from SI samples (n = 5) obtained from cadaveric donors using a mechanical procedure, and separation with immunomagnetic beads coated with anti-EpCAM antibodies. Light and electron microscopy, flow cytometry and immunocytochemistry techniques were used to characterize the isolated cells. Immunohistochemical staining of normal SB biopsies confirmed that the cell cultures maintained an in vivo phenotype as reflected in cytokeratin expression CK18, CK20 and expression of intestine-specific markers such as sucrase isomaltase and maltase glucoamylase. Furthermore, the cells strongly expressed TLR-5, 6, 7, 8 and 10 and several molecules such as CD40, CD86, CD44, ICAM-1 and HLA-DR which are important in triggering cell-mediated immune responses. This novel technique provides a unique in vitro system to study the biology of enterocytes in normal conditions as well as to study inflammatory processes in various small bowel disorders.