Renal tubular HIF-2α expression requires VHL inactivation and causes fibrosis and cysts.

The Hypoxia-inducible transcription Factor (HIF) represents an important adaptive mechanism under hypoxia, whereas sustained activation may also have deleterious effects. HIF activity is determined by the oxygen regulated α-subunits HIF-1α or HIF-2α. Both are regulated by oxygen dependent degradation, which is controlled by the tumor suppressor "von Hippel-Lindau" (VHL), the gatekeeper of renal tubular growth control. HIF appears to play a particular role for the kidney, where renal EPO production, organ preservation from ischemia-reperfusion injury and renal tumorigenesis are prominent examples. Whereas HIF-1α is inducible in physiological renal mouse, rat and human tubular epithelia, HIF-2α is never detected in these cells, in any species. In contrast, distinct early lesions of biallelic VHL inactivation in kidneys of the hereditary VHL syndrome show strong HIF-2α expression. Furthermore, knockout of VHL in the mouse tubular apparatus enables HIF-2α expression. Continuous transgenic expression of HIF-2α by the Ksp-Cadherin promotor leads to renal fibrosis and insufficiency, next to multiple renal cysts. In conclusion, VHL appears to specifically repress HIF-2α in renal epithelia. Unphysiological expression of HIF-2α in tubular epithelia has deleterious effects. Our data are compatible with dedifferentiation of renal epithelial cells by sustained HIF-2α expression. However, HIF-2α overexpression alone is insufficient to induce tumors. Thus, our data bear implications for renal tumorigenesis, epithelial differentiation and renal repair mechanisms

HIF-1α and HIF-2α expression in mouse kidney.

<p>The physiological expression pattern of HIF-1α and HIF-2α was analyzed by immunohistochemistry on kidney sections of normoxic and hypoxic (6 h, 7% O₂) mice. HIF-1α expression was detected in the nuclei of renal tubular epithelial cells after hypoxic stimulation, whereas HIF-2α accumulated in interstitial and glomerular cells.</p

Generation of Ksp/<i>tm</i>HIF-2α.HA transgenic mice.

<p>A. Schematic representation of the pcKsp/<i>tm</i>HIF-2α.HA expression vector used for pronucleus injection consisting of a 1.3 kb Ksp-promoter fragment and an HA-tagged mutated HIF-2α triple mutant cDNA (Ksp, kidney specific; tm, triple mutant; UTR, untranslated region; HA-tag, influenza hemagglutinin epitope tag; poly-A site, poly-adenylation site). Injection of the Ksp/<i>tm</i>HIF-2α.HA construct successfully produced transgenic mice. Two of these were chosen and bred into homozygous strains, which was confirmed by genotyping for Ksp/<i>tm</i>HIF-2α integration and referred as Ksp/<i>tm</i>HIF-2α strain 33 and strain 39, respectively (data not shown). B. Analysis by RT-PCR detected transgenic <i>tm</i>HIF-2α.HA mRNA expression only in whole kidney RNA extracts from mice of strain 39. C. <i>tm</i>HIF-2α.HA protein expression was analyzed in whole kidney extracts from both strains by immunoblot. In parallel to the RNA expression, transgenic <i>tm</i>HIF-2α.HA protein was only expressed in strain 39. Based on these data strain 39 has been termed as <i>tm</i>HIF-2α.HA(+), whereas mice from strain 33 have been defined as <i>tm</i>HIF-2α.HA (−) and served as control strain, having the transgene integrated but not expressed. D. The localization of <i>tm</i>HIF-2α.HA in the mouse kidney was next analyzed by immunohistochemistry against HIF-2α and the HA-tag of the transgene on consecutive sections. Transgenic <i>tm</i>HIF-2α.HA expression was detected in tubular epithelial cells for HIF-2α (left hand panels) as well the HA-tag (right hand panels). E. Kidneys of the <i>tm</i>HIF-2α.HA(−) control strain were negative for both antibodies.</p

Expression of HIF-1α and HIF-2α in the human kidney.

<p>HIF-1α and HIF-2α expression was analyzed by immunohistochemistry in human kidneys. After carbon monoxide (CO) intoxication HIF-1α accumulation was detected in tubular epithelial cells (A) and HIF-2α was detected in interstitial cells and in the glomeruli, indicated by arrows (B). In kidneys of RCC patients HIF-1α and HIF-2α were detected in the tumor tissue as well as in the adjacent kidney (C and D). In the latter HIF-1α was found in tubular epithelial cells (E) and HIF-2α restricted to interstitial cells (F).</p

<i>tm</i>HIF-2α.HA(+) mice develop renal fibrosis and have impaired renal function.

<p>A. Representative photographs of the whole kidney from 15 month old <i>tm</i>HIF-2α.HA transgenic mice. The <i>tm</i>HIF-2α.HA(−) kidneys show a smooth surface, whereas the <i>tm</i>HIF-2α.HA(+) kidneys have an irregular surface structure. B. Immunohistological staining against collagen I shows increased interstitial deposition in close proximity to <i>tm</i>HIF-2α.HA expression in the transgenic kidney. C. Renal fibrosis was scored after SiriusRed staining of collagens. Increased fibrosis was detected in the kidneys of <i>tm</i>HIF-2α.HA(+) mice (results represent mean values of analyzed animals per strain with the error bars being standard deviation; <i>tm</i>HIF-2α.HA(−), n = 10; <i>tm</i>HIF-2α.HA(+), n = 11; * indicates p<0.05). D. The mRNA expression of the fibrosis associated gene TGFβ1 was analyzed by quantitative real-time PCR in whole kidney extracts. The expression was clearly up-regulated in the <i>tm</i>HIF-2α.HA(+) mice (results represent mean values with the error bars being standard deviation; n = 5 per strain). E. Renal function was analyzed by measurement of creatinine in the serum of the transgenic mice. <i>tm</i>HIF-2α.HA(+) mice have significantly increased plasma creatinine levels (* indicates p<0.05).</p

De-repression of HIF-2α in type II lesions of VHL disease kidneys.

<p>Early lesions of human VHL disease kidneys were stained for markers of epithelial dedifferentiation and HIF activation. A. Type II lesions are characterized by reduced E-cadherin expression, no expression of CAIX and little or no staining of HIF-1α. In contrast, strong staining for vimentin, Glut1 and HIF-2α can be seen. B. Type II lesions show pronounced upregulation of the HIF-2α target cyclin D1.</p