S100 calcium-binding protein 16 (S100A16) is implicated in both chronic kidney disease (CKD) and acute kidney injury (AKI). Previous research has shown that S100A16 contributes to AKI by facilitating the ubiquitylation and degradation of glycogen synthase kinase 3ß (GSK3ß) and casein kinase 1α (CK1α) through the activation of HMG-CoA reductase degradation protein 1 (HRD1). However, the mechanisms governing S100A16-induced HRD1 activation and the upregulation of S100A16 expression in renal injury are not fully understood. In this study, we observed elevated expression of Hypoxia-inducible Factor 1-alpha (HIF-1α) in the kidneys of mice subjected to ischemia-reperfusion injury (IRI). S100A16 deletion attenuated the increased HIF-1α expression induced by IRI. Using a S100A16 knockout rat renal tubular epithelial cell line (NRK-52E cells), we found that S100A16 knockout effectively mitigated apoptosis during hypoxic reoxygenation (H/R) and cell injury induced by TGF-ß1. Our results revealed that H/R injuries increased both protein and mRNA levels of HIF-1α and HRD1 in renal tubular cells. S100A16 knockout reversed the expressions of HIF-1α and HRD1 under H/R conditions. Conversely, S100A16 overexpression in NRK-52E cells elevated HIF-1α and HRD1 levels. HIF-1α overexpression increased HRD1 and ß-catenin while decreasing GSK-3ß. HIF-1α inhibition restored HRD1 and ß-catenin upregulation and GSK-3ß downregulation by cellular H/R injury. Notably, Chromatin immunoprecipitation (ChIP) and luciferase reporter assays demonstrated HIF-1α binding signals on the HRD1 promoter, and luciferase reporter gene assays confirmed HIF-1α's transcriptional regulation of HRD1. Additionally, we identified Transcription Factor AP-2 Beta (TFAP2B) as the upregulator of S100A16. ChIP and luciferase reporter assays confirmed TFAP2B as a transcription factor for S100A16. In summary, this study identifies TFAP2B as the transcription factor for S100A16 and demonstrates HIF-1α regulation of HRD1 transcription within the S100A16-HRD1-GSK3ß/CK1α pathway during renal hypoxia injury. These findings provide crucial insights into the molecular mechanisms of kidney injury, offering potential avenues for therapeutic intervention.
Glycogen Synthase Kinase 3 beta , Hypoxia-Inducible Factor 1, alpha Subunit , Animals , Glycogen Synthase Kinase 3 beta/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Mice , Rats , S100 Proteins/metabolism , S100 Proteins/genetics , Reperfusion Injury/metabolism , Reperfusion Injury/genetics , Reperfusion Injury/pathology , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Signal Transduction , Male , Acute Kidney Injury/metabolism , Acute Kidney Injury/pathology , Acute Kidney Injury/genetics , Mice, Inbred C57BL , Kidney/metabolism , Kidney/pathology , Apoptosis , Cell Line , Cell Hypoxia , Mice, Knockout
Recent studies have indicated that the development of acute and chronic kidney disease including renal fibrosis is associated with endoplasmic reticulum (ER) stress. S100 calcium-binding protein 16 (S100A16) as a novel member of the S100 family is involved in kidney disease; however, few studies have examined fibrotic kidneys for a relationship between S100A16 and ER stress. In our previous study, we identified GRP78 as a protein partner of S100A16 in HK-2 cells. Here, we confirmed a physical interaction between GRP78 and S100A16 in HK-2 cells and a markedly increased expression of GRP78 in the kidneys of unilateral ureteral occlusion mice. S100A16 overexpression in HK-2 cells by infection with Lenti-S100A16 also induced upregulation of ER stress markers, including GRP78, p-IRE1α, and XBP1s. Immunofluorescence staining demonstrated that the interaction between S100A16 and GRP78 predominantly occurred in the ER of control HK-2 cells. By contrast, HK-2 cells overexpressing S100A16 showed colocalization of S100A16 and GRP78 mainly in the cytoplasm. Pretreatment with BAPTA-AM, a calcium chelator, blunted the upregulation of renal fibrosis genes and ER stress markers induced by S100A16 overexpression in HK-2 cells and suppressed the cytoplasmic colocalization of GRP78 and S100A16. Co-immunoprecipitation studies suggested a competitive binding between S100A16 and IRE1α with GRP78 in HK-2 cells. Taken together, our findings demonstrate a significant increase in S100A16 expression in the cytoplasm following renal injury. GRP78 then moves into the cytoplasm and binds with S100A16 to promote the release of IRE1α. The subsequent phosphorylation of IRE1α then leads to XBP1 splicing that activates ER stress.
Endoplasmic Reticulum Chaperone BiP/metabolism , Endoplasmic Reticulum Stress , Endoribonucleases/metabolism , Kidney/pathology , S100 Proteins/metabolism , Signal Transduction , X-Box Binding Protein 1/metabolism , Animals , Calcium/metabolism , Cell Line , Fibrosis , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Models, Biological , Protein Binding , Transforming Growth Factor beta1 , Up-Regulation , Ureteral Obstruction/metabolism , Ureteral Obstruction/pathology
Many ion channels participate in controlling insulin synthesis and secretion of pancreatic ß-cells. Epithelial sodium channel (ENaC) expressed in human pancreatic tissue, but the biological role of ENaC in pancreatic ß-cells is still unclear. Here, we applied the CRISPR/Cas9 gene editing technique to knockout α-ENaC gene in a murine pancreatic ß-cell line (MIN6 cell). Four single-guide RNA (sgRNA) sites were designed for the exons of α-ENaC. The sgRNA1 and sgRNA3 with the higher activity were constructed and co-transfected into MIN6 cells. Through processing a series of experiment flow included drug screening, cloning, and sequencing, the α-ENaC gene-knockout (α-ENaC-/-) in MIN6 cells were obtained. Compared with the wild-type MIN6 cells, the cell viability and insulin content were significantly increased in α-ENaC-/- MIN6 cells. Therefore, α-ENaC-/- MIN6 cells generated by CRISPR/Cas9 technology added an effective tool to study the biological function of α-ENaC in pancreatic ß-cells.
