Figure 1.
HIF-1α and HIF-2α expression in mouse kidney.
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.
Figure 2.
Expression of HIF-1α and HIF-2α in the human kidney.
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).
Figure 3.
VHL knockout in mice releases tubular HIF-2α expression.
HIF-1α and HIF-2α expression was analyzed by immunohistochemistry in the kidney cortex (upper panels) and medulla (lower panels) of inducible triple transgenic Pax8-VHL−/− mice after 3 days of doxycyclin treatment (0.2 mg/ml in drinking water). HIF-1α as well as HIF-2α expression appears in renal tubular epithelial cells.
Figure 4.
De-repression of HIF-2α in type II lesions of VHL disease kidneys.
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.
Table 1.
Immunohistochemical labeling of type I and II foci in human VHL kidneys.
Figure 5.
Generation of Ksp/tmHIF-2α.HA transgenic mice.
A. Schematic representation of the pcKsp/tmHIF-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/tmHIF-2α.HA construct successfully produced transgenic mice. Two of these were chosen and bred into homozygous strains, which was confirmed by genotyping for Ksp/tmHIF-2α integration and referred as Ksp/tmHIF-2α strain 33 and strain 39, respectively (data not shown). B. Analysis by RT-PCR detected transgenic tmHIF-2α.HA mRNA expression only in whole kidney RNA extracts from mice of strain 39. C. tmHIF-2α.HA protein expression was analyzed in whole kidney extracts from both strains by immunoblot. In parallel to the RNA expression, transgenic tmHIF-2α.HA protein was only expressed in strain 39. Based on these data strain 39 has been termed as tmHIF-2α.HA(+), whereas mice from strain 33 have been defined as tmHIF-2α.HA (−) and served as control strain, having the transgene integrated but not expressed. D. The localization of tmHIF-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 tmHIF-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 tmHIF-2α.HA(−) control strain were negative for both antibodies.
Figure 6.
tmHIF-2α.HA(+) mice develop renal fibrosis and have impaired renal function.
A. Representative photographs of the whole kidney from 15 month old tmHIF-2α.HA transgenic mice. The tmHIF-2α.HA(−) kidneys show a smooth surface, whereas the tmHIF-2α.HA(+) kidneys have an irregular surface structure. B. Immunohistological staining against collagen I shows increased interstitial deposition in close proximity to tmHIF-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 tmHIF-2α.HA(+) mice (results represent mean values of analyzed animals per strain with the error bars being standard deviation; tmHIF-2α.HA(−), n = 10; tmHIF-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 tmHIF-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. tmHIF-2α.HA(+) mice have significantly increased plasma creatinine levels (* indicates p<0.05).
Figure 7.
Renal cyst development in aged tmHIF-2α.HA(+) mice.
12 month and older tmHIF-2α.HA(+) mice develop multiple renal cysts, mainly in the kidney cortex. These partly form around glomeruli, where the glomerular tuft can be seen (arrows in A). Other cysts arise from distal tubular segments, where transgenic tmHIF-2α.HA expression can be detected in the epithelial lining of the cysts by immunohistochemistry against the HA-tag (arrows in C). In contrast, the glomerular cysts do not show transgene expression, with occasional positive labelling of tubular segments in the vicinity (D).