Table 1.
Physiological measurements in the experimental groups of the rats.
Figure 1.
Necrosis of the renal cortex in 2K1C rats and preservation of the renal tissue in HPP593-treated 2K1C rats (H&E and PAS staining).
In 2K1C rats, renal architecture is preserved, in some cortical areas where injury has progressed dystrophic calcifications are present (asterisk). Necrotic tubular cells retain their cellular outlines, however their nuclei are lost due to ongoing karyolysis. In the clipped kidneys of HPP593-treated 2K1C rats some renal tubules appear to be intact (arrow). In tubules with dedifferentiated epithelium the lumen is narrowed and basement membranes are multilayered (arrowhead). Other tubules are dilated and filled with proteinaceous cast. Interstitium contains lymphoid infiltrate (star).
Figure 2.
Renal tissue fibrosis in 2K1C and HPP593-treated 2K1C rats (Sirius Red staining).
In control kidneys, collagen-specific Sirius Red staining under bright light is observed around glomeruli, basement membrane and blood vessels. Under polarized light, collagen III fibers (yellow-green color) are visible only around blood vessels. In the cortex of 2K1C rats fibrillary collagen I (orange/red color under polarized light) is present in the interstitium and in arteriolar walls. Renal tubules are replaced by the scar tissue in the cortex and at the cortico-medullary junction. Note the sclerotic and crowded glomeruli scattered in the scar tissue of untreated 2k1C rats. In kidneys of HPP593-trearted rats some glomeruli remain crowded (circled in black). Cortical tissue in HPP593-treated 2K1C rats contains large amounts of collagenous matrix as indicated by the intense red color present on these renal sections under bright light. However, unlike in the untreated 2K1C rats, fibrillary collagens I and III are present around blood vessels only (arrows).
Figure 3.
Quantitative analysis of glomerular size (A) and total amounts of fibrous collagens I and III in kidneys of three studied groups.
**p<0.001 vs control.
Figure 4.
Apoptotic cell death in kidneys of 2K1C and HPP593-treated 2K1C rats (TUNEL staining).
Apoptotic cells are absent in the control kidneys. In untreated 2K1C rats, apoptotic cells are present in the cortical scar (arrows with unbroken lines) and in some glomeruli (arrows with broken lines) located close to the cortical capsule. In contrast, in HPP593-treated 2K1C rats apoptotic cells are absent from the glomeruli and present in (arrowheads) or around small atrophic tubules (arrows).
Figure 5.
Renal tissue necrosis is associated with an increase in total and mitochondrial content of BNIP3 protein.
A-immunohistochemical analysis of BNIP3 expression in kidneys. ln control kidneys BNIP3 is expressed in some tubules in the corticomedullary junction. In untreated 2K1C kidneys all necrotic tubules show strong immunoreactivity for BNIP3 protein however there are likely two subpopulations of tubules with different levels of BNIP3 expression. Glomeruli possess a weak immunoreativity for BNIP3. In kidneys of HPP593-treated 2K1C rats BNIP3 positive staining is present in some tubules. B- western blotting analysis of BNIP3 expression. Representative immunoblots and densitometry analysis of BNIP3 expression (n = 6) in total and mitochondrial renal extracts and mitochondrial proteins (n = 10) in the mitochondrial fractions. *p<0.05 and **p<0.001 vs control, ##p<0.001 vs 2K1C.
Figure 6.
Oxidative stress in kidneys of 2K1C rats.
A- Immunohistochemical staining of kidney sections with antibody against 8-HOG. B- quantitative analysis of 8-HOG staining in the kidneys. *p<0.05 and **p<0.001 vs control, ##p<0.001 vs 2K1C.
Figure 7.
Cytosolic and nuclear levels of the key oxidative defense transcription factors NRF2/NRF1.
Representative immunoblots and densitometry analysis of NRF2 and KEAP1 in the cytosolic fraction (A) and NRF1 and NRF2 in the nuclear fraction (B) of renal extracts (n = 6). *p<0.05 and **p<0.001 vs control.
Figure 8.
Mitochondrial mass, protein and DNA levels in clipped kidneys of untreated and HPP593-treated 2K1C rats.
A- representative western blots and quantitative analysis of mitochondrial protein expression in mitochondrial fractions (n = 16), **p<0.001 vs control and 2K1C+HPP593. B-mitochondrial mass estimated as total weight of mitochondria extracted from 1 mg of wet renal tissue (n = 5), **p<0.001 vs control and 2K1C+HPP593. C- mitochondrial DNA copy number per nuclear DNA estimated by copy number of mitochondrial genes COXII and Cytochrom B nuclear actin gene (n≥3), **p<0.001 vs control.
Figure 9.
Autophagy impairment and mitochondrial biogenesis in HPP593-treated and untreated clipped kidneys of 2K1C rats.
Representative immunoblots and densitometry analysis of the levels of key autophagy proteins and PGC-1α in clipped kidneys of 2K1C rats (n = 6). *p<0.05 and **p<0.001 vs control, ##p<0.001 vs 2K1C.
Figure 10.
Mitochondrial function in the clipped kidneys of HPP593-treated 2K1C.
Representative immunoblots and densitometry analysis of cytoplasmic pAMPK/AMPK expression (A) and p62 and Parkin proteins (B) in cytosolic and mitochondrial fractions (n = 6). *p<0.05 and **p<0.001 vs control.
Figure 11.
Vascular endothelial growth factor (VEGF) in the clipped kidneys of 2K1C untreated and HPP593 treated rats.
Representative immunoblot and densitometry analysis of VEGF expression in kidneys of rats (n = 5). **p<0.01 vs control, #p<0.05 vs 2K1C.