Confocal immunofluorescence images were obtained with a NIKON TE2000 microscope with Auto DeBlur deconvolution software as previously described (49). vivo. KIM-1 was directly responsible for phagocytosis in cultured primary rat tubule epithelial cells and also porcine and canine epithelial cell lines. KIM-1 was able to specifically recognize apoptotic cell surface-specific epitopes phosphatidylserine, and oxidized lipoproteins, expressed by apoptotic tubular epithelial cells. Thus, KIM-1 is the first nonmyeloid phosphatidylserine receptor identified to our knowledge that transforms epithelial cells into semiprofessional phagocytes. Introduction Epithelial structures in different organs perform diverse and complex tasks but display stereotyped responses to injury. The kidney epithelium is particularly susceptible to injury due to the character of its blood supply and its ability to concentrate many toxins (1). Injury is characterized by functional deficiencies in handling of salts and water, inability to excrete metabolic toxins, and an innate inflammatory response (2, 3). The damaged segment of the nephron can be remodeled, leading to complete functional recovery, and as such represents a general model of epithelial remodeling after injury. Removal of apoptotic cells and necrotic debris is essential for repair of the tissue with restoration of function (4). Removal of apoptotic cells in a timely fashion has been identified to be a fundamental component of developmental remodeling, regulation of appropriate immune response, and tissue homeostasis (5). It is critical that this process be rapid and efficient to avoid the occurrence of secondary (postapoptotic) necrosis that leads to membrane disruption and leakage of proinflammatory intracellular contents into the tissue. Furthermore, the phagocytic process itself may lead to production of antiinflammatory cytokines URB597 (6). Little is known of the process of clearance of apoptotic cells by epithelial cells or the receptors that may be used by these cells (4, 7, 8). Macrophages, professional phagocytes, are rarely seen in the injured epithelial tubule lumen. The goal of these studies was to determine whether kidney injury moleculeC1 (KIM-1) plays a role in the phagocytic process in epithelial kidney tubule. KIM-1, also known as TIM-1 Mouse monoclonal to CD49d.K49 reacts with a-4 integrin chain, which is expressed as a heterodimer with either of b1 (CD29) or b7. The a4b1 integrin (VLA-4) is present on lymphocytes, monocytes, thymocytes, NK cells, dendritic cells, erythroblastic precursor but absent on normal red blood cells, platelets and neutrophils. The a4b1 integrin mediated binding to VCAM-1 (CD106) and the CS-1 region of fibronectin. CD49d is involved in multiple inflammatory responses through the regulation of lymphocyte migration and T cell activation; CD49d also is essential for the differentiation and traffic of hematopoietic stem cells (T cell immunoglobulin mucin domainsC1), as it is expressed at low levels by subpopulations URB597 of triggered T cells, and hepatitis A disease cellular receptorC1 (HAVCR-1), indicated by hepatocytes, is definitely a transmembrane protein with extracellular mucin and immunoglobulin domains. KIM-1 is not detectable in the normal human being and rodent kidney but is definitely increased in manifestation more than some URB597 other protein in the hurt kidney and is localized mainly to the apical membrane of the surviving proximal epithelial cells (9). KIM-1 is also indicated by dedifferentiated epithelial cells of renal cell carcinomas in humans (10). In this study, we recognized in vivo that apoptotic and necrotic cells of the hurt tubule lumen were phagocytosed by URB597 surviving epithelial cells that communicate Kim-1. Furthermore, Kim-1 itself colocalized to the site of internalization of apoptotic cells. Kim-1 confers on epithelial cells the properties of highly phagocytic cells (or semiprofessional phagocytes) and mediates this by binding specifically to phosphatidylserine (PS) and oxidized lipid epitopes within the apoptotic cell surface. Results Kim-1Cexpressing tubule epithelial cells bind and internalize apoptotic body and necrotic debris in hurt kidneys. Using specific antiCKim-1 antibodies (9), we localized Kim-1 directly adjacent to apoptotic cells and necrotic debris in the lumens of rat kidney tubules in vivo. Kim-1 also surrounds phagocytosed apoptotic body within tubule cells in rat kidney 24 and 48 hours after the kidney had been subjected to ischemic injury (Number ?(Number1,1, A and B). Confocal images (Number ?(Number1C)1C) confirm internalization of apoptotic bodies within Kim-1Cexpressing epithelial cells. You will find phagocytic cups within the apical surface of tubular cells that are lined with Kim-1 (Number ?(Number1C).1C). Proximal tubules in the outer medulla of the kidney, where injury after ischemia is definitely maximal, were obtained for colocalization of Kim-1 staining and the presence of apoptotic body (confirmed by TUNEL staining). While 34.6% 11.8% of Kim-1Cpositive tubules contained cell-internalized apoptotic cells, only 9.4% 3.7% of Kim-1Cnegative tubules contained apoptotic cells (Number ?(Figure1D).1D). TUNEL-positive nuclei were present in intracellular phagosomes and adherent to the luminal surface of Kim-1Cexpressing tubules (Number ?(Figure1E).1E). Importantly, although macrophages, and to a lesser degree additional leukocytes, are recruited to the interstitium of the hurt kidney, they may be hardly ever seen within the tubule lumen, where many apoptotic and necrotic cells are seen (Number ?(Number1F)1F) (11). Therefore, Kim-1Cexpressing kidney epithelial cells avidly phagocytose apoptotic and necrotic cells. Notice also that Kim-1Cexpressing cells lack the macrophage marker CD68 and macrophages in the kidney do not express Kim-1 (Number ?(Figure1F). 1F). Open in a separate window Number 1 Kim-1Cexpressing tubule epithelial cells bind URB597 and internalize apoptotic body and necrotic debris in rat kidneys following.