Podocyte expression of hypoxia-inducible factor (HIF)-1 and HIF-2 during glomerular development

The heterodimeric transcription factors, hypoxia-inducible factor (HIF)-1 and HIF-2, are essential for the maintenance of cellular oxygen homeostasis. In response to hypoxia, stabilized HIF-1alpha and HIF-2alpha proteins bind HIF-1beta and initiate expression of genes that alleviate hypoxic stress, including those promoting neovascularization. Both HIF-1 and HIF-2 stimulate transcription of vascular endothelial growth factor (VEGF), a crucial regulator of vascular development. Because VEGF is highly expressed by metanephric podocytes and collecting ducts, developing mouse kidney was examined for the presence and distribution of HIF-1alpha, HIF-2alpha, and HIF-1beta. The expression of HIF-1alpha and HIF-2alpha mRNAs in newborn mouse kidney was confirmed by RT-PCR and Northern blot analysis. By in situ hybridization, HIF-1alpha and HIF-2alpha mRNAs were highly expressed in the nephrogenic zone of newborn kidney cortex and in the medulla. Particularly intense hybridization was found in podocytes of developing glomeruli and in medullary collecting ducts. Both HIF-1 and HIF-2 heterodimers were identified in newborn kidney lysates by immunoprecipitation with HIF-1alpha, HIF-2alpha, and HIF-1beta antibodies and Western blots. Immunofluorescence analysis of the hypoxia marker, pimonidazole, showed that collecting ducts and many developing tubules undergo severe hypoxia in developing kidney. Immunohistochemistry of newborn kidney demonstrated widespread expression of HIF-1beta protein in nuclei of glomeruli and all tubular segments, whereas HIF-2a protein expression was more restricted and localized chiefly to podocytes of developing glomeruli and developing tubules. HIF-lalpha and HIF-2alpha protein and VEGF mRNA were all strongly induced in embryonic kidneys maintained in hypoxic organ cultures. Collectively, these data suggest that HIF stabilization, by hypoxia and/or by other means, may be critical for VEGF production and kidney vascular development.