CELLULAR APOPTOSIS AND PROLIFERATION IN EXPERIMENTAL RENAL FIBROSIS

Background. The progression of chronic renal failure (CRF) is associat ed with the progressive deletion of renal cells along with the fibrosi s of the kidney. We have studied the role of programmed cell death (ap optosis) in the progression of experimental CRF and renal scarring. Me thods. The sub-total (5/6th) nephrectomy (SNx) model of CRF was studie d in adult male Wister rats, with renal tissue collected from experime ntal and control animals on days 7, 15, 30, 60, 90, and 120 post SNx ( n = 6 per group). These were examined for morphological signs of apopt osis by light and electron microscopy. Further, we stained the nuclear chromatin by the acridine orange fluorescent method and detected sign s of DNA cleavage by endonucleases via the principal of TUNEL staining (ApopTag(TM)). Rates of cellular proliferation were measured simultan eously by immunohistochemical staining for the proliferating cell nucl ear antigen (PCNA). Tn addition, cell division was monitored by counti ng of morphologically mitotic molifs detectable by light microscopy. R esults. Progressive renal insuffciency associated with glomerulosclero sis and tubulointerstitial fibrosis took;;lace in the majority of SNx rats. In these animals, we noted a marked and progressive increase In the number of apoptotic glomerular, tubular as well as interstitial ce lls. The most significant apoptotic changes were seen in the tubules o f remnant kidneys peaking at day 120 post-SNx. At this stage, the incr ease in apoptosis compared to controls was 10.33 +/- 2.67 (M +/- SEM) fold For glomerular cells (P less than or equal to 0.006), 26.20 +/- 4 .56 fold for tubular cells (P < 0.0001) and 4.66 +/- 0.81 fold for int erstitial cells (P less than or equal to 0.001). Parallel changes In t he number of PCNA positive renal cells were observed. Maximal PCNA sta ining was seen at day 120 when the increase with respect to controls w as 14.00 +/- 4.93 fold (P less than or equal to 0.05) for glomerular c ells, 60.01 +/- 12.20 fold (P less than or equal to 0.05) for tubular cells and 28.59 +/- 4.45 fold (P less than or equal to 0.05) for inter stitial cells. As expected, the number of cells undergoing division an d detectable by conventional light microscopy was lower at any time po int to those expressing PCNA. We also observed a close correlation bet ween the severity of tubular atrophy and tubulointerstitial fibrosis w ith the rate of tubular apoptosis (r = 0.970, R-2 = 0.941, P less than or equal to 0.001). Conclusions. We have shown a time-dependent incre ase in apoptosis and PCNA antigen positive staining in the sub-total n ephrectomy model of chronic renal failure correlating with the progres sion of renal fibrosis. PCNA staining did not match analysis for mitos is and was considered to overestimate the number of proliferating cell s in the tissue. With this reservation in mind and taking into account the relative time-frames in vivo of apoptosis and proliferation apopt osis potentially outweighs proliferation by a factor of 2-8-fold, when examined over the same time period. Consequently, even small changes in the finite numbers of apoptotic cells become highly significant. Ou r results have shown the definite role of apoptosis within progression of renal damage and highlighted how it may contribute to the progress ion of tubular atrophy and play a role in the pathogenesis of tubulo-i nterstitial scarring.