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 uropathy (COU). Apoptosis is a complex cellular process consisting of multiple steps, each of which is mediated by families of related molecules. These families may include receptor/ligand molecules such as Fas, Fas ligand, tumor necrosis factor receptor-1 (TNFR-1), and TNF-related apoptosis inducing ligand (TRAIL); signal transduction adapter molecules such as Fas-associated death domain (FADD), TNFR-1 associated death domain (TRADD), receptor-interacting protein (RIP), Fas-associated factor (FAF), and Fas-associated phosphatase (FAP); or effector molecules such as caspases. However, the mechanism of tubular cell apoptosis, as well as the pathogenetic relevance of these apoptosis-related molecules in COU, remains poorly understood.


METHODS
Kidneys were harvested from sham-operated control mice and mice with COU created by left ureter ligation sacrificed in groups of three at days 4, 15, 30, and 45. To detect apoptotic tubular and interstitial cells, in situ end labeling of fragmented DNA was performed. To detect the expression of apoptosis-related molecules, ribonuclease protection assay was used with specific antisense RNA probes for Fas, Fas ligand, TNFR-1, TRAIL, FADD, TRADD, RIP, FAF, FAP, and caspase-8. Immunostaining for Fas, Fas ligand, TRAIL, TRADD, RIP, and caspase-8 was also performed. To assess the role of these molecules in COU-associated renal cell apoptosis, the frequencies of apoptotic tubular and interstitial cells were separately quantitated for each experimental time point, and their patterns of variation were correlated with those of apoptosis-related molecules.


RESULTS
The obstructed kidneys displayed increased apoptosis of both tubular and interstitial cells. Tubular cell apoptosis appeared at day 4 after ureter ligation, peaked (fivefold of control) at day 15, and decreased gradually until the end of the experiment. In contrast, interstitial cell apoptosis sustained a progressive increase throughout the experiment. Apoptosis was minimal at all experimental time points for control and contralateral kidneys. Compared with control and contralateral kidneys, the ligated kidneys displayed a dynamic expression of mRNAs for many apoptosis-related molecules, which included an up to threefold increase for Fas, Fas ligand, TNF-R1, TRAIL, TRADD, RIP, and caspase-8, and an up to twofold increase for FADD and 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 45. The rise and fall of mRNAs between days 4 and 30 paralleled a similar fluctuation in tubular cell apoptosis in that period. The subsequent increase of mRNAs was correlated with a continuous rise of interstitial cell apoptosis. We demonstrated a positive immunostaining for Fas and Fas ligand in the tubular cells at early time points as well as in interstitial inflammatory 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.


CONCLUSIONS
The current study documents a dynamic expression of several molecules that are known to mediate the most crucial steps of apoptosis. It implicates these molecules in COU-associated renal cell apoptosis and in the pathogenesis of this condition. It also lays the foundation for interventional studies, including genetic engineering, to evaluate the molecular control of apoptosis associated with COU.
Choi
Y J
YJ
Renal Pathology Laboratory, Department of Pathology, Department of Medicine, The Methodist Hospital and BaylorCollege of Medicine, Houston, Texas, USA.
Baranowska-Daca
E
E
Nguyen
V
V
Koji
T
T
Ballantyne
C M
CM
Sheikh-Hamad
D
D
Suki
W N
WN
Truong
L D
LD
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000235:genetics / Q000378:metabolism; D002908:Chronic Disease; D003066:Coenzyme A Ligases / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D053222:Fas Ligand Protein; D015870:Gene Expression / Q000502:physiology; D047908:Intracellular Signaling Peptides and Proteins; D007684:Kidney Tubules / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D054595:Protein Tyrosine Phosphatase, Non-Receptor Type 13; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; D018124:Receptors, Tumor Necrosis Factor / Q000235:genetics* / Q000378:metabolism; D012260:Ribonucleases; D053199:TNF Receptor-Associated Death Domain Protein; D047990:TNF Receptor-Associated Factor 1; D053221:TNF-Related Apoptosis-Inducing Ligand; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism; D014517:Ureteral Obstruction / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology*; D019014:fas Receptor / Q000235:genetics* / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C505661:FASLG protein, human; C097186:Faf1 protein, mouse; D053222:Fas Ligand Protein; C505658:Fasl protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; D011506:Proteins; D012333:RNA, Messenger; D018124:Receptors, Tumor Necrosis Factor; D053199:TNF Receptor-Associated Death Domain Protein; D047990:TNF Receptor-Associated Factor 1; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; C505654:Tnfsf10 protein, mouse; C481226:Tradd protein, mouse; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins; D014409:Tumor Necrosis Factor-alpha; D019014:fas Receptor; C506249:RIPK1 protein, human; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; C506251:Ripk1 protein, mouse; D012260:Ribonucleases; C516742:PTPN13 protein, human; D054595:Protein Tyrosine Phosphatase, Non-Receptor Type 13; D017027:Protein Tyrosine Phosphatases; C516743:Ptpn13 protein, mouse; C505530:CASP8 protein, human; C506433:CASP9 protein, human; C505526:Casp8 protein, mouse; C506434:Casp9 protein, mouse; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D003066:Coenzyme A Ligases; C085934:acyl-coenzyme A synthetase, E coli