Mechanism of chronic obstructive uropathy: Increased expression of apoptosis-promoting molecules

Background. We have demonstrated that renal tubular and interstitial cells undergo pronounced apoptosis during the course of chronic obstructive uropa thy (COU). Apoptosis is a complex cellular process consisting of multiple s teps, ach of which is mediated by families of related molecules. These fami lies may include receptor/ligand molecules such as Fas, Pas ligand, tumor n ecrosis factor receptor-1 (TNFR-1), and TNF-related apoptosis inducing liga nd (TRAIL); signal transduction adapter molecules such as Pas-associated de ath domain (FADD), TNFR-1 associated death domain (TRADD), receptor-interac ting protein (RIP), Fas-associated factor (FAE), and Fas-associated phospha tase (FAP); or effector molecules such as caspases. However, the mechanism of tubular cell apoptosis, as well as the pathogenetic relevance of these a poptosis-related molecules in COU, remains poorly understood. Methods. Kidneys were harvested from sham-operated control mice and mice wi th COU created by left ureter ligation sacrificed in groups of three at day s 4, 15, 30, and 45. To detect apoptotic tubular and interstitial cells, in situ end labeling of fragmented DNA was performed. To detect the expressio n of apoptosis-related molecules, ribonuclease protection assay was used wi th specific antisense RNA probes for Fas, Fas ligand, TNFR-1, TRAIL, FADD, TRADD, RIP, FAF, FAP, and caspase-8. Immunostaining for Pas, Pas ligand, TR AIL, TRADD, RIP, and caspase-8 was also performed. To assess the role of th ese molecules in COU-associated renal cell apoptosis, the frequencies of ap optotic tubular and interstitial cells were separately quantitated for each experimental time point, and their patterns of variation were correlated w ith those of apoptosis-related molecules. Results. The obstructed kidneys displayed increased apoptosis of both tubul ar and interstitial cells. Tubular cell apoptosis appeared at day 4 after u reter Ligation, peaked (fivefold of control) at day 15, and decreased gradu ally until the end of the experiment. In contrast, interstitial cell apopto sis sustained a progressive increase throughout the experiment. Apoptosis w as minimal at all experimental time points for control and contralateral ki dneys. Compared with control and contralateral kidneys, the ligated kidneys displayed a dynamic expression of mRNAs for many apoptosis-related molecul es, which included an up to threefold increase for Fas, Pas ligand, TNF-R1, TRAIL, TRADD, RIP, and caspase-8, and an up to twofold increase for FADD a nd FAP, but there was little change for FAF. These mRNAs increased between days 4 and 15, decreased until day 30, but then increased again until day 4 5. The rise and fall of mRNAs between days 4 and 30 paralleled a similar fl uctuation in tubular cell apoptosis in that period. The subsequent increase of mRNAs was correlated with a continuous rise of interstitial cell apopto sis. We demonstrated a positive immunostaining for Fas and Fas ligand in th e tubular cells at early time points as well as in interstitial inflammator y cells at later time points. Although increased expression of TRAIL, TRADD , RIP, and caspase-8 was noted in tubular cells, there was no staining for these molecules in interstitial cells. Conclusion. The current study documents a dynamic expression of several mol ecules that are known to mediate the most crucial steps of apoptosis. It im plicates these molecules in COU-associated renal cell apoptosis and in the pathogenesis of this condition. It also lays the foundation for interventio nal studies, including genetic engineering, to evaluate the molecular contr ol of apoptosis associated with COU.