Deletion of von Hippel-Lindau in glomerular podocytes results in glomerular basement membrane thickening, ectopic subepithelial deposition of collagen {alpha}1{alpha}2{alpha}1(IV), expression of neuroglobin, and proteinuria.

Vascular endothelial growth factor, which is critical for blood vessel formation, is regulated by hypoxia inducible transcription factors (HIFs). A component of the E3 ubiquitin ligase complex, von Hippel-Lindau (VHL) facilitates oxygen-dependent polyubiquitination and proteasomal degradation of HIFalpha subunits. Hypothesizing that deletion of podocyte VHL would result in HIFalpha hyperstabilization, we crossed podocin promoter-Cre transgenic mice, which express Cre recombinase in podocytes beginning at the capillary loop stage of glomerular development, with floxed VHL mice. Vascular patterning and glomerular development appeared unaltered in progeny lacking podocyte VHL. However, urinalysis showed increased albumin excretion by 4 weeks when compared with wild-type littermates with several sever cases (>1000 microg/ml). Many glomerular ultrastructural changes were seen in mutants, including focal subendothelial delamination and widespread podocyte foot process broadening, and glomerular basement membranes (GBMs) were significantly thicker in 16-week-old mutants compared with controls. Moreover, immunoelectron microscopy showed ectopic deposition of collagen alpha1alpha2alpha1(IV) in GBM humps beneath podocytes. Significant increases in the number of Ki-67-positive mesangial cells were also found, but glomerular WT1 expression was significantly decreased, signifying podocyte death and/or de-differentiation. Indeed, expression profiling of mutant glomeruli suggested a negative regulatory feedback loop involving the HIFalpha prolyl hydroxylase, Egln3. In addition, the brain oxygen-binding protein, Neuroglobin, was induced in mutant podocytes. We conclude that podocyte VHL is required for normal maintenance of podocytes, GBM composition and ultrastructure, and glomerular barrier properties.

Kidney development and gene expression in the HIF2alpha knockout mouse.

The hypoxia-inducible transcription factor-2 (HIF2), a heterodimer composed of HIF2alpha and HIF1beta subunits, drives expression of genes essential for vascularization, including vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR-2, Flk-1). Here, we used a HIF2alpha/LacZ transgenic mouse to define patterns of HIF2alpha transcription during kidney development and maturation. Our results from embryonic heterozygotes showed HIF2alpha/LacZ expression by apparently all renal endothelial cells. At 4 weeks of age, glomerular mesangial and vascular smooth muscle cells were also positive together with endothelial cells. These expression patterns were confirmed by electron microscopy using Bluo-gal as a beta-galactosidase substrate. Small numbers of glomerular and tubular epithelial cells were also positive at all stages examined. Light and electron microscopic examination of kidneys from HIF2alpha null embryos showed no defects in renal vascular development or nephrogenesis. Similarly, the same amounts of Flk-1 protein were seen on Western blots of kidney extracts from homozygous and heterozygous HIF2alpha mutants. To examine responsiveness of HIF2alpha null kidneys to hypoxia, embryonic day 13.5 metanephroi were cultured in room air or in mild (5% O(2)) hypoxia. For both heterozygous and null samples, VEGF mRNA levels doubled when metanephroi were cultured in mild hypoxia. Anterior chamber grafts of embryonic HIF2alpha knockouts were morphologically indistinguishable from heterozygous grafts. Endothelial markers, platelet endothelial cell adhesion molecule and BsLB4, as well as glomerular epithelial markers, GLEPP1 and WT-1, were all expressed appropriately. Finally, we undertook quantitative real-time polymerase chain reaction of kidneys from HIF2alpha null embryos and wild-type siblings and found no compensatory up-regulation of HIF1alpha or -3alpha. Our results show that, although HIF2alpha was widely transcribed by kidney endothelium and vascular smooth muscle, knockouts displayed no detectable deficits in vessel development or VEGF or Flk-1 expression.

Divergent expression patterns for hypoxia-inducible factor-1beta and aryl hydrocarbon receptor nuclear transporter-2 in developing kidney.

The hypoxia-inducible factors (HIF) are alpha/beta heterodimeric transcription factors of the basic helix-loop-helix-Per-Arnt-Sim (bHLH-PAS) superfamily and are chiefly responsible for cellular adaptation to oxygen deprivation. HIF function relies on the stabilization of the alpha subunit. When oxygen tension falls, HIF-alpha subunits translocate to the nucleus and, upon dimerization with HIF-beta, activate transcription of target genes, including vascular endothelial growth factor, vascular endothelial growth factor receptor-1 and -2, and WT-1, which are vital for kidney development. HIF-beta subunits are stable regardless of oxygen concentration and constitutively translocate to the nucleus. It was shown previously that HIF-1beta protein expression is nearly ubiquitous in newborn kidney and that HIF-1beta dimerizes with either HIF-1alpha or -2alpha. Here it is shown that aryl hydrocarbon receptor nuclear transporter-2 (ARNT2/HIF-2beta) also heterodimerized with HIF-1alpha and -2alpha. ARNT2/HIF-2beta protein was highly expressed in newborn kidney but decreased significantly with age, whereas HIF-1beta levels remained relatively constant. By immunohistochemical analysis, widespread expression of HIF-1beta was observed in developing and mature kidneys. ARNT2/HIF-2beta protein distribution was restricted to distal segments of developing nephrons and in mature kidney was confined specifically to thick ascending limb of Henle's loop. The data presented here suggest that ARNT2/HIF-2beta is required at high levels during nephrogenesis in distal tubules and later exclusively in thick ascending limb. Furthermore, Hypoxyprobe-1 and lotus lectin co-localization studies showed that developing proximal convoluted tubules were the most severely hypoxic nephron segment in immature kidney. Because HIF-2beta protein was not abundantly expressed in this segment, it may not be engaged in mediating responses to severe hypoxia. The differential distribution patterns for HIF-1beta and -2beta suggest divergent roles during kidney development for these highly related bHLH-PAS proteins.

Hypoxia-inducible factors and kidney vascular development.

Among the genes strongly induced by hypoxia-inducible factors (HIF) and highly expressed during kidney microvascular development is vascular endothelial growth factor, which encodes a potent endothelial mitogen and chemoattractant critical for embryonic vasculogenesis and angiogenesis. In developing kidney, glomerular podocytes are particularly rich sources of vascular endothelial growth factor, which probably serves to attract endothelial precursors into vascular clefts of immature glomeruli, promote their mitosis and differentiation into glomerular endothelial cells, and assist with maintenance of their highly differentiated state through maturation. This article summarizes the structure, function, and expression of HIF and discusses HIF target genes expressed during kidney vascular development. Furthermore, it is speculated that different HIF heterodimers are stabilized in different cell populations, which may lead to cell-selective induction of HIF target genes important for renal vasculogenesis/angiogenesis.

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-2alpha protein expression was more restricted and localized chiefly to podocytes of developing glomeruli and developing tubules. HIF-1alpha 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.