A Bioinformatics Approach Identifies Signal Transducer and Activator of Transcription-3 and Checkpoint Kinase 1 as Upstream Regulators of Kidney Injury Molecule-1 after Kidney Injury

Abstract

Kidney injury molecule-1 (KIM-1)/T cell Ig and mucin domain-containing protein-1 (TIM-1) is upregulated more than other proteins after AKI, and it is highly expressed in renal damage of various etiologies. In this capacity, KIM-1/TIM-1 acts as a phosphatidylserine receptor on the surface of injured proximal tubular epithelial cells, mediating phagocytosis of apoptotic cells, and it may also act as a costimulatory molecule for immune cells. Despite recognition of KIM-1 as an important therapeutic target for kidney disease, the regulators of KIM-1 transcription in the kidney remain unknown. Using a bioinformatics approach, we identified upstream regulators of KIM-1 after AKI. In response to tubular injury in rat and human kidneys or oxidant stress in human proximal tubular epithelial cells (HPTECs), KIM-1 expression increased significantly in a manner that corresponded temporally and regionally with increased phosphorylation of checkpoint kinase 1 (Chk1) and STAT3. Both ischemic and oxidant stress resulted in a dramatic increase in reactive oxygen species that phosphorylated and activated Chk1, which subsequently bound to STAT3, phosphorylating it at S727. Furthermore, STAT3 bound to the KIM-1 promoter after ischemic and oxidant stress, and pharmacological or genetic induction of STAT3 in HPTECs increased KIM-1 mRNA and protein levels. Conversely, inhibition of STAT3 using siRNAs or dominant negative mutants reduced KIM-1 expression in a kidney cancer cell line (769-P) that expresses high basal levels of KIM-1. These observations highlight Chk1 and STAT3 as critical upstream regulators of KIM-1 expression after AKI and may suggest novel approaches for therapeutic intervention.

IL-34 mediates acute kidney injury and worsens subsequent chronic kidney disease

“…Kidney tissues were homogenized in RIPA buffer, and 25 μg total protein was used for immunoblotting, as previously described (62). Blots were probed with mouse monoclonal PTP-ζ Ab (3F8; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, Iowa, USA).…”

“…We performed immunoprecipitation, as previously described (62). Briefly, kidney tissues were homogenized in CHAPS buffer (30 mM Tris-Cl pH 7.5, 150 mM NaCl, 1% CHAPS containing protease and phosphatase inhibitor cocktails [Sigma-Aldrich]) and 250 μg total protein incubated with 1 μg of PTP-ζ Ab overnight at 4°C.…”

Pharmacological and genetic depletion of fibrinogen protects from kidney fibrosis

“…For nondenaturing gels, samples were loaded without heating and sample buffer without DTT or ␤-mercaptoethanol was used. Immunoprecipation was performed as previously described (3,4). Protein (500 g) was used for IP, and 25 g was kept as input, the volume was adjusted to 1 ml for immunoprecipation samples, and 5 g rabbit polyclonal Fg or normal rabbit IgG antibody was added and incubated overnight at 4°C.…”

“…STAT3 chromatin immunoprecipation in snap-frozen kidney tissue was performed as previously described (3). The Fg promoter-specific primers used for RT-PCR are shown in Table 1.…”

“…The rat kidney fibroblast cell line NRK-49F and liver carcinoma cell line Hep G2 were obtained from American Type Culture Collection (Manassas, VA) and maintained in DMEM (Cellgro, Manassas, VA) supplemented with 10% FBS (Invitrogen) at 37°C in a 5% CO2 incubator. Cells were treated with 100 ng/ml IL-6 (R&D Systems) and 200 nM dexamethasone (Sigma) and/or 10 M Stattic (Calbiochem, Philadelphia, PA) and incubated for 24 h. Other cells were transfected with pRC/CMV (EV) or pRC/CMVSTAT3 (kind gift from Dr. David A. Frank, Department of Medical Oncology, Dana Farber Cancer Institute, Boston, MA) as previously described (3). Six hours posttransfection, cells were washed and further incubated for 18 h. Cells were then harvested and processed for RNA and protein isolation.…”