Survivin expression in mouse and human adult kidney and tumor. Immunohistochemistry for survivin in perfusion-fixed mouse kidneys or immersion-fixed human kidneys. Sections through the mouse kidney stained for survivin reveals an identical survivin staining pattern to the rat, namely strong expression in the cortical proximal tubules (A and B), with much less expression in distal tubules and glomeruli, which closely resembles the expression in human kidneys (C–H). D: In human kidney, weak expression can also be seen in medullary collecting ducts. The specificity of the main antibody used (AF886) was further evaluated on consecutive sections of human kidney by antibody preabsorption, with (F) or without (E) full-length survivin protein, processing both sections strictly in parallel. E: Preabsorption almost abolished immunoreactivity. To compare survivin expression levels of human kidneys to corresponding renal tumors, patient samples were collected where the border between kidney and tumor is included and analyzed with two independent antibodies (Table 1, see http://ajp.amjpathol.org). As expected, ambient cytoplasmic staining was observed in renal cell carcinoma with both antibodies. However, the signal intensity derived from the adjacent renal tissue, which again originates from proximal tubular cells, was clearly higher than in the cancerous tissues. Original magnifications: ×40 (G and H); ×100 (A, D, E, and F); ×200 (C); ×400 (B).

Survivin localization on the apical membrane. Immunohistochemistry for survivin on human (A), mouse (B), and rat (C) kidney sections and rat small bowel (D–F). A–C: Survivin in the normal adult kidney profoundly localizes to the apical membrane in all species investigated. This finding is most prominent in perfusion-fixed rat kidneys (C), where for technical reasons tissue preservation is best. E: Interestingly, rat intestine epithelial cells, which are similarly involved in transport, also display a predominance of apical staining, although substantial amounts of survivin staining can also be seen in the cytoplasm. F: However, in the basal crypts, where no transport takes place but intensive proliferation occurs, survivin can be seen in the cytoplasm without membranous enhancement. G and H: Immunofluorescence of rat kidney sections using different detection systems than in the conventional immunohistochemistry also confirms the predominant apical staining pattern. 4,6-Diamidino-2-phenylindole staining in the right hand panel marks the nuclei of all cells (I), in the identical position as shown in H. Original magnifications: ×40 (G); ×200 (D); ×400 (A–C, E, F, and H–I).

Influence of hypoxia, infarction, and cisplatin on survivin expression. The influence of different stresses on survivin were studied, which have clinical relevance for injury to the proximal tubular cells and can cause the development of acute renal failure. A: In cell cultures of primary (hPT) or immortalized (HKC-8) human tubular cells 24 hours of hypoxia (H, 1% O₂) or exposure to the chemical inducer of hypoxia-inducible transcription factor dipyridyl (DP, 100 μmol/L) leads to substantial down-regulation of survivin protein, as measured by immunoblotting. B: Treating rats with hypoxia (9% O₂, 8 hours) did not induce a qualitative difference of survivin staining in the kidney as assessed by immunohistochemistry. However, survivin protein in general seemed to be less abundant than under control conditions and more cytoplasmic. C and D: Segmental infarction in the rat kidney again did not show a difference in the staining pattern for survivin. The border between the ischemic/necrotic (left) and vital (right) tissue can be clearly seen in the middle. The ratio of apical to cytoplasmic staining of survivin in ischemic tubular cells seems to change in favor of cytoplasmic localization under tissue injury, yet apical staining is still present. Induction of acute renal failure by cisplatin (E and F) led to a substantial redistribution of cellular localization of survivin, where the apical orientation is completely lost and the protein is exclusively cytoplasmic.

Influence of survivin (knockdown) on apoptosis of primary tubular cells. The influence of RNA interference against survivin on apoptosis of hPT was measured by annexin V binding (A) and caspase 3/7 activity (B). GFP siRNA was used as a control. The error bars are SEM, in which each experiment has been performed on three independent patient samples. In resting cultures, survivin knockdown did not show any effect on apoptosis, with any assay. However, differences were seen when the cells were stressed for 24 hours, with either cisplatin (middle) or in vitro ischemia (right). The latter is a combination of glucose withdrawal and hypoxia of 1% O₂. Both models lead to a substantial increase of caspase 3/7 activity, when compared with untreated cells (B), whereas only cisplatin exposure leads to a marked increase of annexin V binding (A, middle). A clear increase in the apoptotic fraction of cells was seen by survivin knockdown under both cell stresses (A, statistically significant for cisplatin and for in vitro ischemia, *). Using the caspase 3/7 activity assay, the survivin knockdown achieved a further increase only under cisplatin (B, *).

Survivin expression in adult rat kidney. Immunohistochemistry for survivin (red staining) from adult rat perfusion-fixed kidneys. A: Low-power image from the kidney ranging from cortex to the upper papilla emphasizes the strong cortical expression of survivin with gradual decrease toward the papilla. B–E: Cortical sections from the kidney display strong expression of survivin in proximal tubuli. D: Weaker glomerular staining most likely originates from podocytes (see also Supplemental Figure 1 at http://ajp.amjpathol.org). E: Vascular endothelial cells do not show any staining in the healthy kidney. The changeover of outer to inner medulla shows moderate survivin expression in the S3 segments of proximal tubuli (F), which is weaker than the cortical S1 and S2 sections but stronger than the faint staining in medullary and papillary collecting ducts (F and G; see also Supplemental Figure 1 at http://ajp.amjpathol.org). Original magnifications: ×12.5 (A); ×100 (B, F, and H); ×200 (G); and ×400 (C and D).

Validation of renal tubular survivin expression. A: Frozen human kidney sections were processed by laser-assisted microdissection, cutting cortical tubular sections and whole glomeruli, which served as a control. The microdissected material from 10 patients was analyzed for mRNA expression of survivin by semiquantitative real-time PCR. B: The relative gene expression of four representative patients, in which no or very little expression is seen in glomeruli (G), but substantial expression can be seen in tubular extracts (T). C: Primary hPTs of 10 patients were generated and whole cell lysates immunoblotted for survivin. The position of the molecular weight marker (Page-Ruler; Fermentas, St. Leon-Rot, Germany) at 17 kDa is indicated on the right. Extracts from immortalized human proximal tubular cell lines (HKC-8 and HK2) were loaded as control. To verify the expression of survivin in renal tissue, immunoblots were performed from mouse (D) and human (E) kidney lysates. As control for the correct molecular weight, a whole cell extract from the mouse hepatoma cell line Hepa1c4 was loaded next to in vitro-transcribed and -translated (IVTT) recombinant survivin protein, derived from the full-length human cDNA. Unprogrammed lysates of the same system (reticulocyte lysate) were loaded to exclude the existence of unspecific bands in the proximity of survivin mobility. Furthermore, lysates from hPTs treated with control siRNA against the green fluorescent protein (GFP) or survivin have been loaded. Total protein loaded was 20 μg for Hepa1c4, 30 μg for hPT, 100 μg for murine kidneys, and 200 μg for human kidney lysates. C–E: The polyclonal rabbit anti-survivin antibody AF886 was used in all immunoblots.

Survivin expression in developing kidney. Immunohistochemistry for survivin in developing human (A–C) and rat (D–F) kidneys. Surprisingly, human embryonic kidneys and newborn rat kidneys showed staining for survivin only in tubular structures, which is similar to adult tissues. Interestingly, the area of most intense growth and where the most undifferentiated structures can be found, the so-called nephrogenic zone in the outer cortex, shows markedly less intense staining than in the more developed central regions. Whereas developing proximal tubular structures show a high level of survivin expression (C, upper structure), developing collecting ducts do not show any staining (C, lower structure). Similarly survivin is not detectable in developing glomeruli (B, E) or interstitial and endothelial cells. Very low level of staining can be seen in the typical S-shaped bodies of rat kidneys in the nephrogenic zone (F, center). Original magnifications: ×100 (A and D); ×400 (B, C, E, and F).

Subcellular localization and influence of survivin (knockdown) on cell cycle of primary tubular cells. A: Immunofluorescence for survivin on primary hPTs cultured on glass slides (left). A: Right: 4,6-diamidino-2-phenylindole-staining of the identical positions. The top clearly shows the difference in subcellular localization from predominant cytoplasmic in the majority of the population to predominant nuclear in individual cells. The latter may indicate the timely proximity to mitosis. The bottom shows survivin localizing to the mitotic spindle apparatus of a dividing cell. Furthermore, protein expression is much higher in this dividing cell, than in the surrounding cells. Exposure time of top left 1/4 second, of bottom left 1/16 second. B: RNA interference was performed on hPTs, either for GFP as a control or for survivin, which led to a pronounced decrease of protein levels, as measured by immunoblotting. C and D: Twenty-four hours after survivin knockdown in hPTs, measurement of DNA content by using the fluorescence-activated cell sorting-propidium iodide staining method revealed a G₂/M arrest, indicating failure of cells to exit mitosis. The difference of G₂/M fractions, as well as the reduction in G₁ phase, between survivin and GFP knockdown was statistically significant (*). C: The mean values of three independent experiments from different patients, where the error bars are SEM. D: The original results of one representative experiment.