Evidence of Tubular Hypoxia in the Early Phase in the Remnant Kidney Model

Figure 2. Increased pimonidazole uptake by remnant kidneys in the early phase. Double immunohistochemical staining with perfused lectin (brown) and a hypoxic probe, pimonidazole (gray) (×200). (a) No pimonidazole uptake was observed in the renal cortex in the sham group, while tubules in the deep medulla were pimonidazole-positive. (b) In the early phase after nephron loss (4 and 7 d), many tubules in the cortex showed pimonidazole uptake, indicating that these tubules were in a hypoxic condition. Treatment with an ARB, olmesartan, decreased pimonidazole uptake in the cortex of remnant kidneys at both time points (4 and 7 d), indicating that hypoxia of the tubulointerstitium may be mediated at least in part by activation of RAS. (c) In the extended phase (2 wk) of RK, rats showed sustained adduction of pimonidazole. (d) A high-magnification picture (×400) of sham animals showed the normal structure of perfused postglomerular capillary networks surrounded by tubules negative for pimonidazole. (e) In contrast, a high-magnification picture of the RK group showed pimonidazole adduction, indicating localized hypoxia.

Figure 3. Upregulation of hypoxia-responsive proteins in remnant kidneys. (a) Imunoprecipitation of HIF-1α using cortical tissues of sham and remnant kidney rats. HIF-1α was undetectable in sham kidneys, but was increased in remnant kidneys. (b) Transcription of hypoxia-responsive genes. Quantitative RT-PCR of the renal cortex of sham and remnant kidneys showed that all genes, including EPO, GLUT-1, and VEGF, were increased in remnant kidneys. Olmesartan-treated animals showed a decrease in all hypoxia-responsive genes. A, P < 0.05 versus sham; B, P < 0.05 versus RK.

Figure 4. Change in pattern of postglomerular peritubular capillaries in remnant kidneys. Immunohistochemical studies were conducted on binding of injected lectin to endothelial cells of functioning capillaries. (a) In sham-operated animals, peritubular capillaries were arranged in a regular pattern with patent capillary lumina. (b) In early remnant kidneys (RK) (representative picture from 4d RK), most capillary lumina were distorted and had relatively narrow lumens. (c) ARB treatment restored the normal capillary morphology (representative picture from 7d RK+ARB). (d) Histogram of luminal area demonstrated that remnant kidneys harbored more narrow peritubular capillaries. Treatment with ARB prevented this change. (e) Correlation between mean capillary luminal area and pimonidazole score in each animal showed that narrowing of capillaries correlated with hypoxia.

Figure 5. Tissue perfusion studies with Hoechest 33342 dye. Tissue perfusion status was assessed by injection of Hoechest 33342 dye exactly 30 s before removal of the kidney. In sham animals, nuclear fluorescence was intense in every area (a), whereas nuclear fluorescence signals in the RK group were distinctly decreased in the tubulointerstitial area (b). Treatment with ARB increased nuclear fluorescence (c), indicating that ARB restored net perfusion in remnant kidneys. Semiquantitative measurement of nuclear luminosity supported these results (Table 3).