Immediately before imaging, the cell-seeded substrate assemblies were mounted individually in 6-cm cell culture plates under standard media

Immediately before imaging, the cell-seeded substrate assemblies were mounted individually in 6-cm cell culture plates under standard media. Traction force microscopy was performed with an automated upright epifluorescence microscope (Labophot 2; Nikon, Melville, NY) equipped with immersion objectives and an environmental chamber. cells from oxidative cell injury to promote cell survival. Kidney failure occurs when the kidneys lose their ability to function because of acute or chronic diseases.1Both acute kidney injury (AKI) and chronic kidney disease (CKD) are major kidney diseases associated with high rates of morbidity and mortality.2Although two distinct entities, emerging evidence strongly indicates close interconnection between AKI and CKD, wherein the occurrence of one strongly predicts the risk of the other.3,4This interconnection also points to the presence of possible common underlying molecular mechanisms in AKI and CKD.4Renal tubular epithelial cells constitute most of the renal mass and are the common damaged cell type in both AKI and CKD.5,6Hypoxia, ischemia reperfusion (IR) injury, and oxidative stress damage are MDM2 Inhibitor common pathologic assaults that inflict injury on epithelial cells, and the endurance of these cells strongly influences the clinical outcome.7,8 Cell adhesion plays a major role in kidney injury and repair. In response to insults such as ischemia or toxins, kidney epithelial cells lose their cellcell and MDM2 Inhibitor cellmatrix interactions, leading to loss of cell polarity, increased permeability, and cell death.911These events contribute to intraluminal aggregation of cells and proteins, causing tubular obstruction.12,13The loss of cell adhesion in injured cells proceeds changes in the distribution of actin and actin-binding proteins with altered structural characteristics and cytoskeletal changes10that lead to reduced sodium transport and other impairments.14Kidney epithelium has a remarkable regenerative capacity after ischemic/toxic injury. During the repair process, kidney tubular epithelial cells undergo a complex series of regenerative events such as proliferation, migration, and epithelialmesenchymal transition, leading to restoration of functional tubular epithelial cells.15Cell adhesion plays a prominent role in these regenerative processes.16 Recently, we identified immunoglobulin (Ig) and proline-rich receptor-1 (IGPR-1) as a novel cell adhesion molecule encoded by transmembrane and Ig domain-containing 2 (TMIGD2).17IGPR-1/TMIGD2 is expressed in human endothelial and epithelial cells but is absent in the mouse genome. IGPR-1 is involved in angiogenesis, and its activity regulates endothelial capillary tube formation and cell migration.17Our further investigation resulted in the identification of TMIGD1, the second family member of IGPR-1. TMIGD1 is predicted to be expressed in humans, mice, and other species. A recent study suggests that TMIGD1 is expressed in normal human colon epithelial cells, and its expression appears to be down-regulated in human colon tumors.18The data presented here suggest that TMIGD1 is expressed in kidney tubular epithelial cells and that it acts to protect kidney epithelial cells from oxidative cell injury. TMIGD1 expression changes in mouse AKI and in deoxy-corticosterone acetate (DOCA) and sodium chloride (DOCA salt)-induced chronic hypertensive kidney disease models, suggesting a possible role for TMIGD1 in kidney cell injury. == Materials and Methods == == Plasmids, shRNA, and Antibodies == A FOXO3 mouse TMIGD1 clone (cDNA clone MGC:74197, MDM2 Inhibitor IMAGE:30311543) was purchased from Open Biosystems (Huntsville, AL) and subsequently was cloned as aMyc(alias c-myc) tag in its C-terminus into retroviral pQCXIP vector via NotI and BamH1 sites.Myc-tagged TMIGD1 was further sequenced to confirm its sequence identity. Rabbit polyclonal anti-TMIGD1 antibody was made against a peptide that corresponded to 20 amino acids in the extracellular domain of TMIGD1. TMIGD1 shRNA (catalog SC 94146-sh) was purchased from Santa Cruz Biotechnology (Dallas, TX). It consists of a pool of three to five lentiviral vector plasmids, each encoding TMIGD1-specific 19 to 25 nt (plus hairpin) shRNAs. == Cell Lines == Human embryonic kidney.