• Jump to main content
• Jump to navigation
• nature.com homepage
• Publications A-Z index
• Browse by subject
• ISN

• My account
• Submit manuscript
• Register
• Subscribe

Login

Cell Biology – Immunology – Pathology

Kidney International (2001) 60, 924–934; doi:10.1046/j.1523-1755.2001.060003924.x

Renal cell apoptosis in chronic obstructive uropathy: The roles of caspases

Luan D Truong, Yeong-Jin Choi, Chun Chui Tsao, Gustavo Ayala, David Sheikh-Hamad, George Nassar and Wadi N Suki

Renal Pathology Laboratory, Department of Pathology, and Department of Medicine, The Methodist Hospital and Baylor College of Medicine, Houston, Texas, USA

Correspondence: Luan D. Truong, M.D., Department of Pathology, M.S. 205, The Methodist Hospital, Houston, Texas 77030, USA. E-mail: ltruong@bcm.tmc.edu

Received 4 October 2000; Revised 14 March 2001; Accepted 16 March 2001.

Abstract

Renal cell apoptosis in chronic obstructive uropathy: The roles of caspases.

Background

Apoptosis of tubular and interstitial cells is well documented in kidneys with chronic obstructive uropathy (COU) and probably plays an important role in the pathogenesis of this condition. The molecular control of apoptosis in COU remains poorly understood. Apoptosis in general is known to proceed initially along distinct pathways, which later converge into a common arm characterized by orderly activation of caspases. Caspases are cytosolic enzymes that belong to a 12-member family and serve as effector molecules for apoptosis. The role of individual caspases in mediating renal cell apoptosis in kidneys with COU is studied.

Methods

Kidneys were harvested from sham-operated mice and mice with COU created by left ureter ligation at days 4, 7, 15, 20, and 30. The following studies were performed: (1) determination of dried kidney weight; (2) in situ end labeling of fragmented DNA to detect apoptotic tubular and interstitial cells; (3) ribonuclease protection assay with specific anti-sense RNA probes for caspases 1, 2, 3, 6, 7, 8, 9, 11, and 12 to detect the expression of individual caspases; (4) immunostaining for caspases; and (5) assay for caspase 3. To assess the role of caspases 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 individual caspases.

Results

The obstructed kidneys showed progressive tissue loss (60% of control at day 15). Apoptosis of both tubular and interstitial cells was seen in obstructed kidneys. Tubular cell apoptosis peaked at four days after ureter ligation (13-fold of control), remained high between days 4 to 15, and thereafter decreased rapidly. Apoptotic interstitial cells were scanty initially, but gradually increased throughout the entire experiment. Apoptosis was minimal throughout the experiment in control and contralateral kidneys. In control and contralateral kidneys, caspases 2, 3, 6, 7, 8, and 9 mRNAs were expressed at low levels, whereas those for caspases 1, 11, and 12 were not detected. The obstructed kidneys displayed increased expression of all tested caspases. Caspases 1, 11, and 12 mRNAs were detected in obstructed kidneys in a common pattern characterized by a sharp increase at day 4, followed by a decrease until day 20, and a subsequent sharp increase until the end of the study at day 30. A similar pattern was noted for other caspases (2, 3, 6, 7, 8, and 9), which maximally reached twofold to fourfold that of controls. Immunostaining for caspases 1, 2, 3, 6, 7, 8, and 9 showed the same pattern characterized by focal and weak expression in proximal tubules of control or contralateral kidney, contrasting with increased staining in atrophic or dilated tubules of obstructed kidneys. Interstitial cells also displayed staining for several caspases, which paralleled the increasing density of interstitial cells toward the end of the experiment. Caspase-3 assay showed a marked increased activity in obstructed kidneys that reached fourfold and sevenfold of control at days 4 and 30, respectively. The rise and fall of caspase mRNAs between days 4 and 30 paralleled a similar fluctuation in tubular cell apoptosis. The subsequent increase of mRNAs was correlated with a continuous rise of interstitial cell apoptosis.

Conclusions

Urinary obstruction in mice induces apoptosis of both tubular and interstitial cells in the affected kidney in a distinctive pattern that parallels an increased expression of caspases. This correlation suggests that these caspases mediate COU-associated renal cell apoptosis. Among the evaluated caspases, increased renal caspase 3 activity implies its central role in renal cell apoptosis associated with urinary obstruction.

Keywords:

tubular cell apoptosis, interstitial cell apoptosis, cell death, fibrosis, kidney injury, urinary obstruction, cysteinyl aspartate-specific proteinase

Urinary obstruction results in a constellation of renal parenchymal changes collectively called chronic obstructive uropathy (COU). These changes characteristically include tubular atrophy, interstitial fibrosis, interstitial inflammation, and renal tissue loss, whereas glomeruli or blood vessels show no significant injury^1,2,3,4. More recently, it has been demonstrated that urinary obstruction faithfully induces progressive apoptosis of both renal tubular and interstitial cells. This process may be pathogenetically important since tubular cell apoptosis is known as a major factor responsible for the progressive renal tissue loss in kidneys with COU and interstitial cell apoptosis is probably relevant to the renal interstitial injury in this condition⁵. Understanding the molecular control of renal cell apoptosis in COU should not only help elucidate its pathogenesis, but also might be pertinent to the pathogenesis of chronic renal tubulointerstitial injury in general^6,7.

Although the molecular control of apoptosis is only partially known and may show subtle tissue-specific variations, a general common theme is well accepted. Accordingly, a large variety of apoptosis inducers, classified into two broad categories, that is, extracellular environmental stimulation and cell or organelle membrane changes, can initiate apoptotic signals. These signals are transduced through cytosol by an ever-increasing number of mediator molecules that belong to distinct families, each of which mediates a specific apoptotic pathway^8,9. These pathways, however, converge into a common arm characterized by an orderly activation of caspases, which serve as effector molecules for apoptosis^{10,11,12,13,14}. Caspases (cysteinyl aspartate-specific proteinase) are cytosolic enzymes that belong to a family with 14 members (caspases 1 through 14), 12 of which are found in mammals¹³. These enzymes share common structural motifs and a predilection to cleave their substrates after an aspartate residue. Caspases exist in latent forms (procaspases) and require activation, which occurs in an orderly "cascade" manner to become functional. Three general classes of caspases are recognized, that is, the initiators, the executioners, and the cytokine processors. The initiators (caspases 2, 8, 9, and 10), situated at the upstream of the caspase cascade, can be individually activated by many apoptotic sensors of different death pathways and, once activated, can initiate the caspase cascade by activating the downstream executioners (caspases 3, 6, and 7). These executioner caspases can cleave numerous cellular substrates, including structural proteins and cellular enzymes needed for cellular homeostasis, leading to the structural changes of apoptosis^{10,11,12,13,14}. Less is known about the function of the cytokine processors (caspases 1, 4, 5, 11, and 12); they may promote not only apoptosis but cytokine functions as well¹².

The role of caspases as the mediators of renal tubulointerstitial cell injury has been explored only recently, and most pertinent data are still preliminary and are derived from cell cultures. Increased expression of some caspases was noted in apoptotic renal tubular cells induced by cis-platinum^15,16,17,18, staurosporine (a protein kinase C inhibitor)¹⁵, cyclosporine A^19,20, hypoxia^21,22, human immunodeficiency virus²³, ischemia/reperfusion²⁴, cytoplasmic injection of cytochrome C^25,26, reactive oxygen species²⁷, and loss of contact with extracellular matrix²⁸. Caspases also were implicated in interstitial cell apoptosis in polycystic mouse kidneys²⁹.

Recognizing the central role of caspases in apoptosis and a lack of knowledge about the molecular control of apoptosis in obstructed kidneys, we have attempted to evaluate the relevance of individual caspases in this process. For this purpose, tubular and interstitial cell apoptosis was quantitated in mouse kidneys with COU induced by unilateral ureter ligation, and the findings were correlated with the renal expression of caspases.

METHODS

Experimental design

Under inhalation anesthesia with methoxyflurane, C57B16 male mice (Harlan Animal Farm, Houston, TX, USA) weighing 25 to 35 grams were subjected to complete ligation of the left ureter at the ureteropelvic junction using double silk suture. Animals were subsequently placed on a regular diet and sacrificed at days 4, 7, 15, 20, and 30. These time points were chosen because a pilot study showed these mice to span the entire quantitative spectrum of tubular and interstitial cell apoptosis. Three to six mice were used for each time point. For the controls, a group of six mice were sham operated and sacrificed at day 0.

Dried kidney weight

Dried kidney weight, which is used as an indicator of renal tissue loss, was determined as follows. Each kidney was weighed immediately after harvesting. The ratio of dried:wet kidney weight for each kidney was derived by weighing a representative coronal section of that kidney at the time of harvesting and after thorough desiccation. The whole kidney dried weight, calculated from this ratio and the whole kidney wet weight, was expressed as the percentage of body weight determined at the time of sacrifice.

Tissue preparation

Portions of the control, ligated, and contralateral kidneys were fixed in 10% buffered formalin or frozen in embedding media (OCT compound; Sakura Finetek, Torrance, CA, USA) and cut in 4 micron sections for routine histology and in situ end labeling of fragmented DNA or frozen-section immunohistochemistry. The remaining kidney tissues from animals of the same experimental duration were pooled together and snap frozen in liquid nitrogen at -70°C for subsequent RNA extraction.

Detection and quantitation of tubular and interstitial cell apoptosis by in situ end labeling of fragmented DNAs

For the detection of renal cell apoptosis, in situ end labeling of fragmented DNA was performed as previously detailed^3,30. In situ end labeling of fragmented DNA can detect both apoptotic and necrotic cells. Therefore, light microscopic criteria for identifying apoptotic cells, that is, individual cells with hyperchromatic nuclear fragments and condensed but preserved cytoplasm, were used during the examination of the tissue sections submitted to in situ labeling. These criteria also were used to confirm the presence of apoptotic cells in the routine light microscopic tissue sections consecutive to those used for in situ labeling. Tubular cells undergoing apoptosis were quantitated under a 10 objective lens of a Nikon microscope, by counting all apoptotic tubular cells within 5 to 10 random fields in either the cortex or medulla. The frequency of apoptotic tubular cells was expressed as a percentage of the total tubular cells in the same fields. The frequency of interstitial cell apoptosis also was similarly quantitated. Although both interstitial fibroblasts and interstitial inflammatory cells may undergo apoptosis, it is not possible to differentiate precisely these two cell types when they become apoptotic; therefore, separate quantitation for them was not done.

Ribonuclease protection assay

Ribonuclease protection assay was used to detect and quantitate caspase mRNAs in kidney tissue. In this type of assay, a cocktail of probes is used to detect the mRNAs of several preselected functionally related molecules simultaneously³¹. The assay was performed as follows. For each time point, kidney tissue from three to six animals were pooled together, and total RNA was isolated from the control, ligated, and contralateral kidneys using the RNAzol-B method (Tel-Test, Friendswood, TX, USA). Subsequently, the ribonuclease protection assay was performed using the RiboQuant RNase Protection Assay Kit (Pharmingen, San Diego, CA, USA). This kit contained cDNA templates for caspases 1, 2, 3, 6, 7, 8, 11, and 12 and cDNA template for ribosomal protein L32 as an internal control. A separate cDNA template for caspase 9, custom designed by Pharmingen, also was used for the ribonuclease protection assay. Labeled antisense RNA probes were synthesized from these cDNA templates, using [-³²P] UTP in an in vitro transcription reaction, and hybridized for 16 hours at 56°C with 10 g of total RNA extracted from the control, ligated, and contralateral kidneys. The hybridized products were digested with a mixture containing ribonuclease and proteinase K. During this procedure, the unhybridized RNAs and the free RNA probes were digested, but the hybridized RNAs were protected from digestion (ribonuclease protection assay). The hybridized RNAs were heat denatured and electrophoresed on polyacrylamide gel for two hours at 50 W and 50°C. The gel was then dried and exposed to x-ray film at -70°C. The resultant bands were scanned and quantitated using PhotoShop and UTHSCSA software. Band intensity was normalized to that of L32 in the same reaction.

Immunohistochemical staining

Immunostaining, using the antibodies detailed in the Table 1, was performed to assess the expression and the localization of caspases. The monospecificity of these antibodies was demonstrated by Western blotting. Two antibodies were used to stain for caspase 9. One antibody (p10) reacts with a fragment of the caspase 9 molecule, which encompasses the amino acids 315 to 397 and helps detect both the precursor and the active subunit, and the other antibody (p35) reacts with the 100 to 270 fragment and should preferentially detect the precursor. A modified avidin-biotin-peroxidase technique with tyramide enhancement (Dako, Carpinteria, CA, USA) was applied to snap-frozen kidney tissue sections from control, contralateral, and ligated kidneys. The controls included replacement of the primary antibodies by non-immune serum or neutralization of the primary antibodies with the corresponding caspase blocking peptides (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Table 1 - Antibodies against caspases.

Assay of caspase 3 activity

Since caspase 3 is well-known as the principal executioner caspase^9,10, it was selected for assay by a colorimetric assay kit (Biovision, Palo Alto, CA, USA). In this assay, caspase 3 cleaves the chromophore p-nitroanilide (pNA) from the labeled substrate DEVD-pNA; the caspase 3 activity is determined by the concentration of pNa measured spectrophotometrically at 400 nm. The test was performed as follows: 10 mg of kidney tissue from control, contralateral, or ligated kidneys were homogenized and left in 300 L of cell lysis buffer at 4°C for 15 minutes. After centrifugation at 10,000 g for five minutes, the supernatant, which contained cytoxolic extraction, was harvested and determined for protein concentration by the Lowry method. After an adjustment to a concentration of 200 g/50 L, 50 L of the supernatant were added to 50 L of reaction buffer and 5 L of 4 mmol/L DEVD-pNA . The mixture was incubated at 37°C for two hours and subjected to spectrophotometric reading by a microtiter plate reader set at a wavelength of 405 nm.

RESULTS

Pathologic findings

The obstructed kidneys developed progressive tubulointerstitial changes, whereas the glomeruli and blood vessels remained normal throughout the experimental period. The tubular changes included atrophy, dilation, and simplification of the tubular epithelium and tubular cell apoptosis; the interstitial changes included fibrosis, inflammatory cell infiltration, fibroblast proliferation, and apoptosis of interstitial cells. No significant changes were noted in the control and contralateral kidneys Figure 1.

Figure 1.

Light microscopy. (A) A contralateral kidney at day 7 does not show any significant changes. (B) The corresponding obstructed kidney shows tubular atrophy, interstitial expansion, and interstitial inflammation (periodic acid-Schiff, 1200).

Dried kidney weight

Progressive loss of renal tissue was observed in obstructed kidneys, with the renal weight at day 15 decreasing to approximately 60% of control Figure 2. The weight of the contralateral kidneys gradually increased throughout the experimental course.

Figure 2.

Dried kidney weight. Although the dried weight of the contralateral kidneys increased, the dried weight of the obstructed kidneys gradually decreased as the result of renal tissue loss. Symbols are: () ligated kidneys; () contralateral kidneys.

Apoptosis

Although rare apoptotic tubular or interstitial cells were noted in routine tissue sections, accurate identification and quantitation of apoptotic cells were greatly facilitated by in situ end labeling for fragmented DNA Figure 3. Tubular cell apoptosis peaked four days after ureter ligation (13-fold of control), remained high between days 4 through 15, and thereafter decreased rapidly Figure 4a. Apoptotic interstitial cells were less numerous than apoptotic tubular cells in the initial phase of the study, but gradually increased throughout the entire experimental period Figure 4b. Although it was not possible to differentiate the interstitial fibroblasts from inflammatory cells that underwent apoptosis accurately, the latter were probably more numerous, especially toward the end of the experiment. Apoptotic cells were rarely seen in the glomeruli and virtually not present in control or contralateral kidneys throughout the experiment.

Figure 3.

Renal cell apoptosis. (A) Only a rare apoptotic tubular cell, detected by positive in situ end labeling for fragmented DNA, is noted in a contralateral kidney at day 7. This pattern is also observed in the control kidney. (B) The corresponding obstructed kidney displays pronounced tubular cell apoptosis. A few apoptotic interstitial cells also are seen (in situ labeling with methyl green/alcian blue counterstain, 1200).

Figure 4.

Frequency of apoptotic cells. (A) The frequency of tubular cell apoptosis in obstructed kidneys peaks at day 4 and decreases gradually to the level of contralateral kidneys. (B) The frequency of apoptotic interstitial cells in obstructed kidneys increases gradually throughout the experimental duration. Symbols are: () ligated kidneys; () contralateral kidneys.

RNase protection assay for caspase mRNA

The original blot is shown in Figure 5a, and the spectrophotometric quantitation of individual caspases is shown in Figure 6. In control kidneys, caspases 8, 3, 6, 2, and 7 mRNAs were expressed at low levels, whereas mRNAs for caspases 1, 11, and 12 were not detected. The expression in contralateral kidneys was similar to that of control kidneys. The obstructed kidneys displayed increased expression of all tested caspases in a dynamic fashion that reflected the duration of ureter ligation Figure 6. Caspase 1, 11, and 12 mRNAs were all detected in obstructed kidneys, in a common pattern characterized by a sharp increase at day 4, followed by a decrease until day 20, and a subsequent sharp increase until the end of the study at day 30. A similar pattern was noted for other caspases (2, 3, 6, 7, and 8), which maximally reached twofold to fourfold of control. The patterns of caspase 9 mRNA expression in the control, contralateral, and obstructed kidneys were similar to those of other caspases, except that a peak level was sustained between days 4 and 7 Figure 7.

Figure 5.

Ribonuclease protection assay. The ribonuclease protection assay blot demonstrates a low level of mRNAs of caspases 8, 3, 6, 2, and 7 in control (Cont) or contralateral (CL) kidneys at day 7. The obstructed kidneys at 4, 7, 15, 20, and 30 days (4L, 7L, 15L, 20L, 30L) show increased mRNAs of caspases 8, 3, 6, 2, and 7 and neoexpression of mRNAs of caspases 11, 12, and 1.

Figure 6.

Expression pattern of individual caspases. The individually illustrated blots are taken from the composite blot shown in Figure 5. The band density is measured spectrophotometrically and plotted against the experimental duration.

Figure 7.

Ribonuclease protection assay for caspase 9. There is a low level of caspase 9 mRNA control (Cont) or contralateral (CL) kidneys at day 7. The obstructed kidneys at 4, 7, 15, 20, and 30 days (4L, 7L, 15L, 20L, 30L) show a pattern of caspase 9 expression similar to those of other caspases.

Immunohistochemistry

Immunostaining for caspases 1, 2, 3, 6, 7, and 8, using antibodies with specificity for both the precursors and the active subunits of individual caspases, showed a similar pattern for all tested caspases Figure 8. The staining specificity was supported by a negative result when the primary antibodies were replaced by nonimmune serum or preabsorbed by blocking peptides. The control and contralateral kidneys displayed focal staining for caspases 2, 3, 6, 7, and 8 in proximal tubules, but interstitial cells were not stained. In the obstructed kidney, atrophic or dilated tubules in both cortex and medulla showed increased caspase staining for all caspases, with an increased expression being equally noted in tubular cells with or without features of apoptosis. The staining was most pronounced between days 4 and 15 but persisted to the end of the study. The interstitial cells also displayed staining for caspases 3, 6, and 7, which paralleled the increasing density of interstitial cells toward the end of the experiment. Immunostaining for caspase 9 with two different antibodies Table 1 showed that the precursor was localized predominantly to normal proximal tubular cells. On the other hand, the atrophic tubules in obstructed kidneys showed only weak staining for the precursor, but more strongly expressed the active subunit Figure 9. These observations suggest that caspase 9 is located in renal tubules, becomes activated during urinary obstruction, and may play a role in tubular cell apoptosis.

Figure 8.

Immunostaining for caspases 3 and 8. A control kidney shows a weak expression of caspase 3 in proximal tubules (A), but a negative result is noted for the medulla (B). An obstructed kidney at day 7 shows a strong expression of caspase 3 in both cortical and medullary tubules (C and D). The staining is noted in both atrophic and dilated (*) tubules. An obstructed kidney at day 30 shows a strong expression of caspase 8 in atrophic tubules in both cortex and medulla (E and F). The stain is located not only to the tubular cells (open arrows), but also to the interstitial cells (arrowheads; avidin-biotin peroxidase, with tyramide enhancement, 600 for all panels).

Figure 9.

Immunostaining for caspase 9. A control kidney shows a weak expression of caspase 9 in proximal tubules (A). The atrophic tubules in an obstructed kidney at day 7 display weak staining for the precursor of caspase 9 (B), but show stronger expression of the active subunit of caspase 9 (C).

Caspase 3 activity assay

Caspase 3 activity in control and contralateral kidneys was equally low (0.06 and 0.07; Figure 10). This activity was increased in ligated kidneys throughout the experiment, and reached between sixfold and sevenfold at days 4 and 30, respectively.

Figure 10.

Caspase 3 activity. The caspase 3 activity is measured by the level of the chromophore p-nitroanilide released from the caspase 3-specific substrate DEVD.

DISCUSSION

Kidneys with urinary obstruction in both humans and experimental animals develop progressive tubulointerstitial injury^1,2,3,4,5. Among the different elements of this injury, tubular cell apoptosis has recently emerged as a crucial lesion that may be pathogenetically related to other tubular changes and be responsible for the profound renal tissue loss seen in obstructed kidneys^1,3. Interstitial changes, including fibrosis, inflammatory cell infiltrate, and vascular remodeling, also develop in kidney with urinary obstruction and have been shown to correlate with interstitial cell apoptosis³. These observations strongly imply a disturbance of tubular and interstitial cell cycles, including an increased apoptosis of these cells, in the pathogenesis of COU.

Understanding the molecular control of COU-associated renal cell apoptosis is not only pathogenetically relevant, but also promises novel treatments for COU, whereby the focus is on retarding renal injury to allow time for restoring the urinary tract patency. Renal changes collectively called chronic tubulointerstitial injury can occur in practically any renal disease in advanced stages and are well accepted as a major determinant of renal outcome. These changes are practically identical to those in obstructed kidneys, including progressive renal cell apoptosis^3,4,6,7. Understanding how cells in obstructed kidneys develop apoptosis, therefore, may have a broad implication. In spite of these considerations, the molecular control of this cellular process in COU is virtually unknown.

Studies, mostly from cell cultures, have suggested that several environmental or cellular changes can induce apoptosis along two separate major pathways, one of which is initiated by activation of cell membrane death receptors and the other by disturbances of the mitochondrial membrane^9,10. These two pathways, however, converge into a common arm characterized by orderly activation of caspases in a cascade manner^9,10. How this simplified scheme of apoptosis is relevant to renal cell apoptosis in COU is not entirely known. Several factors, including hypoxia, ischemia, activated cytokines, growth factors, angiotensin II, and reactive oxygen species, which are known to induce apoptosis in general, develop during the course of COU and may initiate renal cell apoptosis³. Families of molecules that mediate the two aforementioned apoptotic pathways have been shown in kidneys with COU^32,33. However, the role of caspases as the mediators of the final common steps leading to apoptosis in this condition has not been evaluated.

The pattern of COU-associated renal cell apoptosis was well characterized in rats^1,3. Since the cDNA probes for the caspases included in the current study are mouse specific, we have created COU in mice to see whether a distinctive pattern of renal cell apoptosis develops, because if that is the case, the course of apoptosis and caspase expression can be studied simultaneously and correlated. The ribonuclease protection assay was used to study caspase mRNAs. Ribonuclease protection assay, which represents a modification of the traditional Northern hybridization, is an ideal method for simultaneous evaluation of mRNAs of several target molecules³¹. This approach is made possible by a technique that allows for the synthesis of several antisense RNA probes of related molecules by in vitro transcription and the simultaneous use of these probes in the hybridization. It is more sensitive and reliable than Northern hybridization and more quantitative than reverse transcriptase-polymerase chain reaction analysis, since the assay includes both probes for the target molecules and those for "housekeeping" genes, which simultaneously detect the mRNAs of these genes and those of target genes. The former probes serve as internal controls for quantitation of the target mRNAs.

In mouse kidneys with urinary obstruction, we found that tubular cell apoptosis increased rapidly, peaked at day 4, and regressed to the baseline values toward the end of the study. This pattern of variation paralleled those of all evaluated caspases, an observation that implicates increased caspase expression in the development of tubular cell apoptosis. In contrast, apoptosis of interstitial cells exhibited a continuous rise that accelerated toward the end of the experiment and paralleled progressive interstitial injury, including increased interstitial fibroblasts and inflammatory cells. Since this late apoptotic activity coincided precisely with a sharp up-regulation of caspase expression, these two events may be pathogenetically related. Immunostaining with monospecific antibodies with specificities for both the precursors and active forms of most caspases included in this study showed that normal and contralateral kidneys displayed weak caspase expression in some cortical tubules. Strong staining that involved many damaged tubules, however, was noted throughout both cortex and medulla of the kidneys with urinary obstruction. A similar pattern of increased staining also was noted for interstitial cells. Therefore, the immunostaining not only corroborates the mRNA data, but also helps define the tissue localization of caspases. Apoptosis was virtually not seen in control and contralateral kidneys, a finding that correlated with the absence or baseline expression of caspases at both the message and protein levels. The obtained data, taken together, implicate the caspases evaluated in this study as the mediators of both tubular and interstitial cell apoptosis induced by urinary obstruction.

The roles of individual caspases in COU-induced renal cell apoptosis deserve further consideration with regards to their three main general functions: apoptotic execution, apoptotic initiation, and cytokine processing. Caspases 1, 11, and 12, which belong to the interleukin-1 converting enzyme subfamily of proteases and share structural homology, were thought to function mainly as cytokine processors^{13,34,35,36,37}, but recent studies suggested that these caspases also mediate apoptosis, including that in kidney^1,34,37. Mice with either caspase 1- or 11-null mutation had normal apoptosis but failed to produce and secrete interleukin-1^38,39. In contrast, either caspases 1 or 11 overexpression induced apoptosis of murine fibroblasts, which was attenuated by bcl-2 or crmA, two well-known caspase inhibitors³⁴. Rat renal tubular cell apoptosis induced by either hypoxia or ischemia/reperfusion expressed high levels of caspase 1^17,18. Mice with the caspase 12 null mutation developed normally, but embryonic fibroblasts or mature renal proximal tubular cells from these mutant mice were resistant to endoplasmic reticulum-mediated apoptotic signals³⁷. Our study documented a common pattern of caspase 1, 11, and 12 expression. This pattern was undetectable in normal and contralateral kidneys, but for obstructed kidneys there was a peak at day 4, when tubular cell apoptosis was at its maximum, and another peak at day 30, when tubular cell apoptosis returned to baseline values, but interstitial changes, including lymphocytic infiltration, were pronounced. Taken together, these observations suggest that caspases 1, 11, and 12 are relevant to both apoptosis and interstitial inflammation in kidneys with COU.

Caspases 2, 8, and 9 represent the prototypes of initiator caspases^8,9,10. Caspases 2 and 8 initiate the activation of other downstream caspases in the death receptor pathway, whereas caspase 9 recently was shown to play a similar role in the mitochondrial pathway^8,9,10,40. Caspase-2 overexpression was documented in apoptotic renal tubular cells induced by ischemia/reperfusion²⁴ and was associated with renal interstitial cell apoptosis in murine polycystic kidneys²⁹. Caspase 8 has been implicated in the apoptosis of mouse collecting duct cells induced by a loss of contact with extracellular matrix²⁸. Although caspase 9 has been implicated in the apoptosis of various cell types, including neurons¹², hepatocytes⁴¹ and cardiac myocytes⁴², only two recent reports suggest a role for caspase 9 in renal tubular cell apoptosis observed in experimental hypertensive nephrosclerosis⁴³ or in vitro hypoxia⁴⁴. The current study documents increased expression of caspases 2, 8, and 9 in obstructed kidneys that paralleled the pattern of apoptosis in these kidneys. Activation of caspase 9 also was documented imunohistochemically. These observations implicate both the death receptor and mitochondrial pathways in renal cell apoptosis induced by urinary obstruction. Indeed, in addition to caspases, most molecules known to mediate these two pathways are expressed in kidneys with COU^32,33.

Caspases 3, 6, and 7 share structural homology and are situated most downstream along the caspase cascade^10,11,13. They are activated by the initiator caspases such as caspase 2 or caspase 8 and help execute the apoptotic function by activating cytosolic or nuclear proteolytic enzymes, which mediate structural changes of apoptosis. Among the executioner caspases, caspase 3 is the best studied and represents the central molecule situated at the crossroad of all known apoptotic pathways^{10,11,12,13,14}. Caspase 3-mediated apoptosis has been demonstrated in many cell types, and its null mutation in mice induced an enlarged brain that was lethal in the perinatal period that was apparently due to a failure of the neurons to die during fetal development^14,45,46. Caspase 3 has been implicated in renal tubular cell apoptosis induced by cytochrome c²⁶, cis-platinum, ischemia/reperfusion²⁴, hypoxia-induced adenosine 5'-triphosphate (ATP) depletion^21,22, and cyclosporine A^19,20. It also may involve interstitial cell apoptosis in murine polycystic kidneys. Much less is known about the role of caspases 6 and 7 in renal cell apoptosis. A role for caspases 3 and 7 in mediating apoptosis in kidneys with COU is supported by their increased expression, which maximally reached 2.3-fold of control and reflected the observed course of apoptosis. The pro-apoptotic role of caspase 3 is also supported by the continuous increase of caspase 3 enzymatic activity in obstructed kidneys, that when measured by the level at which caspase 3 cleaves its specific substrate DEVD-pNA, maximally reached tenfold of control. On the other hand, overexpression of caspase 6 in obstructed kidneys was less than impressive, and its place in the scheme of COU-associated renal cell apoptosis remains unclear.

The current study documents a dynamic expression of several caspases in kidneys with urinary obstruction that correlates with a distinctive pattern or renal cell apoptosis. The findings suggest that these caspases, especially caspase 3, may serve as mediators of renal cell apoptosis induced by urinary obstruction, and may be relevant to renal cell apoptosis as a common feature of chronic tubulointerstitial injury in general.

References

1. Gobe, GC & Axelsen, RA: Genesis of renal tubular atrophy in experimental hydronephrosclerosis in the rat: Role of apoptosis. Lab Invest 1987 56:273–281,  | PubMed | ISI | ChemPort |
2. Wilson, D & Klahr, S: Urinary tract obstruction, in Disease of the Kidney 1993, (5th ed), edited by Schrier RW, Gottschalk CW, Boston, Little Brown and Co., pp 657–687
3. Truong, LD, Sheikh-Hamad, D, Chakraborty, S, Suku, WN: Cell apoptosis and proliferation in obstructive uropathy. Semin Nephrol 1998 18:641–651,  | PubMed | ISI | ChemPort |
4. Nagle, RB & Bulger, RE: Unilateral obstructive nephropathy in the rabbit. II. Late morphologic changes. Lab Invest 1978 38:270–278,  | PubMed | ISI | ChemPort |
5. Truong, LD, Petrusevska, G, Yang, G, et al: Cell apoptosis and proliferation in experimental chronic obstructive uropathy. Kidney Int 1996 50:200–207,  | PubMed | ISI | ChemPort |
6. Schainuck, LI, Striker, GE, Cutler, RE, Benditt, EP: Structural-functional correlations in renal disease. II. The correlations. Hum Pathol 1970 4:631–641,
7. D'Amico, G, Ferrario, F, Rastaldi, MP: Tubulointerstitial damage in glomerular diseases: Its role in the progression of renal damage. Am J Kidney Dis 1995 26:124–132,  | PubMed | ChemPort |
8. Pothana, S, Dong, Z, Valery, M, et al: Apoptosis: Definition, mechanisms, and relevance to disease. Am J Med 1999 107:489–506,  | Article | PubMed | ISI | ChemPort |
9. Granville, DJ, Carthy, CM, Hunt, DWC, McManus, BM: Apoptosis: Molecular aspects of cell death and disease. Lab Invest 1998 78:893–913,  | PubMed | ISI | ChemPort |
10. Budihardjo, I, Oliver, H, Lutter, M, et al: Biochemical pathways of caspase activation during apoptosis. Annu Rev Cell Dev Biol 1999 15:269–290,  | Article | PubMed | ISI | ChemPort |
11. Nicholson, DW: Caspase structure, proteolytic substrates, and function during apoptotic cell death. Cell Death Differ 1999 6:1028–1042,  | Article | PubMed | ISI | ChemPort |
12. Zheng, TS, Hunot, S, Kuida, K, Flavell, RA: Caspase knockouts: Matters of life and death. Cell Death Differ 1999 6:1043–1053,  | Article | PubMed | ISI | ChemPort |
13. Cohen, GM: Caspases: The executioners of apoptosis. Biochem J 1997 326:1–16,  | PubMed | ISI | ChemPort |
14. Kumar, S: Mechanisms mediating caspase activation in cell death. Cell Death Differ 1999 6:1060–1066,  | Article | PubMed | ISI | ChemPort |
15. Fukuoka, K, Takeda, M, Kobayashi, M, et al: Distinct interleukin-1-converting enzyme family proteases mediate cisplatin-and staurosporine-induced apoptosis of mouse proximal tubule cells. Life Sci 1998 62:1125–1138,  | Article | PubMed | ISI | ChemPort |
16. Zhan, Y, van de Water, B, Wang, Y, Stevens, JL: The roles of caspase-3 and bcl-2 in chemically-induced apoptosis but not necrosis of renal epithelial cells. Oncogene 1999 18:6505–6512,  | Article | PubMed | ISI | ChemPort |
17. Lau, AH: Apoptosis induced by cisplatin nephrotoxic injury. Kidney Int 1999 56:1295–1298,  | Article | PubMed | ISI | ChemPort |
18. Chen, Z, Seimiya, H, Naito, M, et al: ASK1 mediates apoptotic cell death induced by genotoxic stress. Oncogene 1999 18:173–180,  | Article | PubMed | ISI | ChemPort |
19. Shihab, FS, Andoh, TF, Tanner, AM, et al: Expression of apoptosis regulatory genes in chronic cyclosporine nephrotoxicity favors apoptosis. Kidney Int 1999 56:2147–2159,  | Article | PubMed | ISI | ChemPort |
20. Ortiz, A, Lorz, C, Catalan, M, et al: Cyclosporin A induces apoptosis in murine tubular epithelial cells: Role of caspases. Kidney Int 1998 54(Suppl 68):S25–S29,  | Article | ISI |
21. Edelstein, CL, Shi, Y, Schrier, RW: Role of caspases in hypoxia-induced necrosis of rat renal proximal tubules. J Am Soc Nephrol 1999 10:1940–1949,  | PubMed | ISI | ChemPort |
22. Feldenberg, LR, Thevananther, S, del Rio, M, et al: Partial ATP depletion induces Fas-and caspase-mediated apoptosis in MDCK cells. Am J Physiol 1999 276:F837–F846,  | PubMed | ISI | ChemPort |
23. Conaldi, PG, Biancone, L, Bottelli, A, et al: HIV-1 kills renal tubular epithelial cells in vitro by triggering an apoptotic pathway involving caspase activation and Fas upregulation. J Clin Invest 1998 102:2041–2049,  | PubMed | ISI | ChemPort |
24. Kaushal, GP, Singh, AB, Shah, SV: Identification of gene family of caspases in rat kidney and altered expression in ischemia-reperfusion injury. Am J Physiol 1998 274:F587–F592,  | PubMed | ISI | ChemPort |
25. Li, F, Srinivasan, A, Wang, Y, et al: Cell-specific induction of apoptosis by microinjection of cytochrome c: Bcl-xL has activity independent of cytochrome c. J Biol Chem 1997 272:30299–30305,  | Article | PubMed | ISI | ChemPort |
26. Chang, SH, Phelps, PC, Berezesky, IK, et al: Studies on the mechanisms and kinetics of apoptosis induced by microinjection of cytochrome c in rat kidney tubule epithelial cells (NRK-52E). Am J Pathol 2000 156:637–649,  | PubMed | ISI | ChemPort |
27. Lieberthal, W, Triaca, V, Koh, JS, et al: Role of superoxide in apoptosis induced by growth factor withdrawal. Am J Physiol 1998 275:F691–F702,  | PubMed | ISI | ChemPort |
28. Park, MY, Lee, RH, Lee, SH, Jung, JS: Apoptosis induced by inhibition of contact with extracellular matrix in mouse collecting duct cells. Nephron 1999 83:341–351,  | Article | PubMed | ISI | ChemPort |
29. Ali, SM, Wong, VY, Kikly, K, et al: Apoptosis in polycystic kidney disease: Involvement of caspases. Am J Physiol 2000 278:R763–R769,  | ISI | ChemPort |
30. Gavrieli, Y, Sherman, Y, Ben-Sasson, SA: Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 1992 119:493–501,  | Article | PubMed | ISI | ChemPort |
31. GILMAN, M: Ribonuclease protection assay, in Current Protocols in Molecular Biology 1993 (vol 1), edited by AUSUBEL FM, BRENT R, KINGSTON RE, et al New York, John Wiley and Sons, Inc., pp 4.7.1–4.7.8,
32. Choi, Y-J, Baranowska-Daca, E, Nguyen, V, et al: Mechanism of chronic obstructive uropathy: Increased expression of apoptosis-promoting molecules. Kidney Int 2000 58:1481–1491,  | Article | PubMed | ISI | ChemPort |
33. Choi, Y-J, Mendoza, L, Rha, S-J, et al: The role of P53-dependent activation of caspases in chronic obstructive uropathy: Evidence from P53 null mutant mice. J Am Soc Nephrol 2001 12:983–992,  | PubMed | ISI | ChemPort |
34. Miura, M, Zhu, H, Rotello, R, et al: Induction of apoptosis in fibroblasts by IL-1-converting enzyme, a mammalian homolog of the C. elegans cell death gene ced-3. Cell 1993 75:653–660,  | Article | PubMed | ISI | ChemPort |
35. Wang, S, Miura, M, Jung, YK, et al: Identification and characterization of Ich-3, a member of the interleukin-1 converting enzyme (ICE)/Ced-3 family and an upstream regulator of ICE. J Biol Chem 1996 271:20580–20587,  | Article | PubMed | ISI | ChemPort |
36. Schotte, P, Criekinge, WV, Van de Craen, M, et al: Cathepsin B-mediated activation of proinflammatory caspase-11. Biochem Biophys Res Commun 1998 251:379–387,  | Article | PubMed | ISI | ChemPort |
37. Nakagawa, T, Zhu, H, Morishima, N, et al: Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-. Nature 2000 403:98–103,  | Article | PubMed | ISI | ChemPort |
38. Kuida, K, Lippke, JA, Ku, G, et al: Altered cytokine export and apoptosis in mice deficient in interleukin-1 converting enzyme. Science 1995 267:2000–2002,  | PubMed | ISI | ChemPort |
39. Wang, S, Muira, M, Jung, Y-K, et al: Murine caspase-11, an ICE-interacting protease, is essential for the activation of ICE. Cell 1998 92:501–509,  | Article | PubMed | ISI | ChemPort |
40. Li, P, Budihardjo, I, Srinivasula, SM, et al: Cytochrome c and dATP-dependent formation of Apaf-1/caspase 9 complex initiates an apoptotic cascade. Cell 1997 91:479–489,  | Article | PubMed | ISI | ChemPort |
41. Ozoren, N, Kim, K, Burns, TF, et al: The caspase 9 inhibitor Z-LEHD-FMK protects human liver cells while permitting death of cancer cells exposed to tumor necrosis factor-related apoptosis-inducing ligand. Cancer Res 2000 60:6259–6265,  | PubMed | ISI | ChemPort |
42. Zhu, H, McElwee-Witmer, S, Perrone, M, et al: Phenelephryl protects neonatal rat cardiomyocytes from hypoxia and serum deprivation-induced apoptosis. Cell Death Differ 2000 7:773–784,  | Article | PubMed | ISI | ChemPort |
43. Ying, WZ & Sanders, PW: Cytochrome c mediates apoptosis in hypertensive nephrosclerosis in Dahl/ Rapp rats. Kidney Int 2001 59:662–672,  | Article | PubMed | ISI | ChemPort |
44. Dong, Z, Saikuma, P, Patel, Y, et al: Serine protease inhibitors suppress cytochrome c-mediated caspase 9 activation and apoptosis during hypoxia-reoxygenation. Biochem J 2000 347:669–677,  | Article | PubMed | ISI | ChemPort |
45. Kuda, K, Zheng, TS, Na, S, et al: Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice. Nature 1996 384:368–372,  | Article |
46. Woo, M, Hakem, R, Soengas, MS, et al: Essential contribution of caspase-3/CPP32 to apoptosis and its associated nuclear changes. Genes Dev 1998 12:806–819,  | PubMed | ISI | ChemPort |

Main navigation

ISN resources

• Nephrology Gateway
• Chinese edition
• Spanish edition
• Portuguese edition
• Contact us
• Society news

NPG resources

• KI Japanese home page
• Nature Reviews Nephrology
• Nature Reviews Urology

NPG Journals

by Subject Area

• Chemistry

  • Chemistry
  • Drug discovery
  • Biotechnology
  • Materials
  • Methods & Protocols

• Clinical Practice & Research

  • Cancer
  • Cardiovascular medicine
  • Dentistry
  • Endocrinology
  • Gastroenterology & Hepatology
  • Methods & Protocols
  • Pathology & Pathobiology
  • Urology

• Earth & Environment

  • Earth sciences
  • Evolution & Ecology

• Life sciences

  • Biotechnology
  • Cancer
  • Development
  • Drug discovery
  • Evolution & Ecology
  • Genetics
  • Immunology
  • Medical research
  • Methods & Protocols
  • Microbiology
  • Molecular cell biology
  • Neuroscience
  • Pharmacology
  • Systems biology

• Physical sciences

  • Physics
  • Materials

by A - Z Index

Extra navigation

ARTICLE NAVIGATION - FULL TEXT

• Table of contents
• Download PDF
• Send to a friend
• Rights and permissions
• Order Commercial Reprints
• CrossRef lists 19 articles citing this article
• Scopus lists 47 articles citing this article
• Save this link
• Abstract
• METHODS
• RESULTS
• DISCUSSION
• References
• Figures and Tables

• Export citation
• Export references

• Papers by Truong

Top

• Kidney International
• ISSN: 0085-2538
• EISSN: 1523-1755
• © 2010 International Society of Nephrology

• About NPG
• Contact NPG
• RSS web feeds
• Help

• Privacy policy
• Legal notice
• Accessibility statement
• Terms

• Nature News
• Nature jobs
• Nature Asia
• Nature Education

• partner of AGORA, HINARI, OARE, INASP, CrossRef and COUNTER