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

Negative regulation of CD8 expression via Cd8 enhancer–mediated recruitment of the zinc finger protein MAZR

Coreceptor expression is tightly regulated during thymocyte development. Deletion of specific Cd8 enhancers leads to variegated expression of CD8αβ heterodimers in double-positive (DP) thymocytes. Here we show CD8 variegation was correlated with an epigenetic “off” state, linking Cd8 enhancer function with chromatin remodeling of the adjacent genes Cd8a and Cd8b1 (Cd8). We show the zinc finger protein MAZR, encoded by the Zfp278 gene, bound the Cd8 enhancer and interacted with the corepressor N-CoR complex in double-negative thymocytes. MAZR was down-regulated in DP and single-positive CD8(+) thymocytes. Enforced expression of MAZR led to impaired Cd8 activation and variegated CD8 expression. Our results demonstrate epigenetic control of the Cd8 gene loci and identify MAZR as an important regulator of Cd8 gene expression