Hypoxia-Inducible Factor-1[alpha] Activates the Transforming Growth Factor-[beta]/SMAD3 Pathway in Kidney Tubular Epithelial Cells

Background: Kidney injury, including chronic kidney disease and acute kidney injury, is a worldwide health problem. Hypoxia and transforming growth factor-[beta] (TGF-[beta]) are well-known factors that promote kidney injury. Hypoxia-inducible factor (HIF) and SMAD3 are their main downstream transcriptional factors. Hypoxia-HIF pathway and TGF-[beta]/SMAD3 pathway play a crucial role in the progression of kidney injury. However, reports on their interactions are limited, and the global transcriptional regulation under their control is almost unknown. Methods: Kidney tubular epithelial cells were cultured and stimulated by hypoxia and TGF-[beta]. We detected global binding sites of HIF-1[alpha] and SMAD3 in cells using chromatin immunoprecipitation-sequencing (ChIP-Seq), and measured the gene expression using RNA-sequencing (RNA-Seq). ChIP-quantitative PCR (qPCR) was used to quantitatively evaluate bindings of SMAD3. Results: ChIP-Seq revealed that 2,065 and 5,003 sites were bound by HIF-1[alpha] and SMAD3, respectively, with 614 sites co-occupied by both factors. RNA-Seq showed that hypoxia and TGF-[beta] stimulation causes synergistic upregulation of 249 genes, including collagen type I alpha 1 (COL1A1) and serpin peptidase inhibitor, clade E, member 1, which are well-known to be involved in fibrosis. Ontology of the 249 genes implied that the interaction of HIF-1[alpha] and SMAD3 is related to biological processes such as fibrosis. ChIP-qPCR of SMAD3 at HIF-1[alpha] binding sites near COL1A1 and SERPINE1 indicated that HIF-1[alpha] promotes the bindings of SMAD3, which is induced by TGF-[beta]. Conclusions: These findings suggest that HIF-1[alpha] induced by hypoxia activates the TGF-[beta]/SMAD3 pathway. This mechanism may promote kidney injury, especially by upregulating genes related to fibrosis. Keywords: Chromatin immunoprecipitation-sequencing, Hypoxia-inducible factor-1[alpha], Hypoxia, Kidney tubular epithelial cell, RNA-sequencing, SMAD3, Transforming growth factor-[beta]