Histopathology and apoptosis in an animal model of reversible renal injury

Abstract

High adenine phosphate (HAP) diet serves as an animal model of chronic renal failure (RF). Induction of RF and establishment of end organ damage require long exposure periods to this diet. Previously, we have shown that RF is reversible after diet cessation even after protracted administration. In this study, we explored the underlying renal changes and cellular pathways occurring during administration and after cessation of the diet. Kidneys were obtained from rats fed HAP diet for 7 weeks, and from rats fed HAP diet followed a 10 week recovery period on normal diet. The kidneys of HAP diet group were significantly enlarged due to tubular injury characterized by massive cystic dilatation and crystal deposition. Kidney injury was associated with markers of apoptosis as well as with activation of apoptosis related pathways. Diet cessation was associated with a significant reduction in kidney size, tubules diameter, and crystals deposition. The recovery from renal injury was coupled with regression of apoptotic features. This is the first study showing the potential reversibility of long standing RF model, allowing optimal evaluation of uremia-chronic effects.

Fig. 1

Macroscopic morphology of kidneys. Macroscopic morphology of kidneys obtained from 3 groups: 7-weeks adenine-treated, after reversibility and control. The HAP diet induced a significant increase in the size of the kidney due to the cystic dilatation of the tubules. After reversibility the kidney size was decreased and its surface became irregular.

Fig. 2

Histological evaluation of kidneys. Sections A and B were obtained from kidney of HAP diet treated rat; A – mid-longitudinal section of the kidney with extensive nephropathy area (i.e., 70–80% of tissue area – grade 5). B – two typical areas not involved by nephropathy are delineated. These areas are characterized by tubules lined by cells having normal eosinophilic-stained cytoplasm. Sections C and D were obtained from kidney of rat treated by HAP diet and then undergoing 10-week reversibility period; C – mid-longitudinal section demonstrating relatively reduced nephropathy area compared with A (i.e., up to 40% of the tissue area – grade 3). D – two typical areas not involved by nephropathy are delineated. These areas are characterized by tubule lined by cells having normal – eosinophilic-stained cytoplasm. In the areas of nephropathy, the brownish crystals were still present in the tubular lumen, as seen in the HAP diet treated rats. Note: for comparative purposes, photos A and B, and C and D, have the same respective magnification (×2).

Fig. 3

Apoptosis assessment in kidneys. Apoptosis was assessed using two different methods: 3A – TUNEL staining (original magnification 50) demonstrating a significantly higher level of DNA fragmentation (orange) in HAP diet kidneys. Only scattered apoptotic cells were found in the control and reversibility kidneys. 3B – Western blot analysis and graphic presentation for cleaved caspase 3 and caspase 3 protein levels in control (C), HAP diet (HAP) and reversibility (RE) groups. The analysis demonstrates a significantly higher ratio of cleaved caspase 3 to caspase 3 in HAP diet kidneys. Both methods suggest that apoptosis plays an important part the pathogenesis of this model of RF.