Ureter , the Guardian Angel of Nephron : The Impact of Pelvicalyceal System on Apoptotic Activity in an Experimental Obstructive Uropathy Model

Objective: We aimed to assess the impact of obstructive uropathy and its revision on renal apoptotic and inflammatory activity in an experimental model in rabbits. Methods: Twenty-four rabbits were separated into four different groups. Ureteropelvic junction (UPJ) obstruction model was performed in Group 1 and 2. Ureterovesical junction (UVJ) obstruction model was performed in Group 3 and 4. Thirty days after the operations, bilateral nephrectomy was performed in Group 1 and 3. Thirty days after the initial surgery, the obstruction was revised in Group 2 and 4. Thirty days after the revision surgeries, bilateral nephrectomy was performed in Group 2 and 4. The apoptotic and inflammatory activity was measured at the protein level and nephrectomy specimens were examined histologically. Results: Caspase 3 levels were significantly higher in operated left kidneys than the levels obtained in the right counterparts. The increase in procaspase 9 and caspase 3 activities in the UVJ obstruction group was less than those of the UPJ obstruction group (p<0.05, p<0.01, respectively). After the revision of obstruction, the decrease in procaspase 9 and caspase 3 activities was more significant in Group 4 than in Group 2 (p<0.05, p<0.01, respectively). Cyclooxygenase-2 expression decreased insignificantly in Group 2 and 4 when compared to Group 1 and 3 (p>0.05, p>0.05, respectively). Conclusion: The rabbits with a UVJ obstruction had lower apoptotic indexes as compared to those with a UPJ obstruction. The apoptotic activity decreased to almost normal levels following an adequate revision of the obstruction in both groups, but this improvement was more significant in animals with a UVJ obstruction.


INTRODUCTION
Physiological turnover of the cell including both cell proliferation and programmed cell death is essential for preserving the functional integrity of the kidney.Obstructive uropathy (OU), a major cause of chronic renal failure in childhood, is characterized by the decreased cell proliferation and increased apoptosis (1).The ureteropelvic junction (UPJ) and ureterovesical junction (UVJ) obstruction are among the most common known causes of the obstructive uropathy in children (2).Both the pelvicalyceal system and the ureter are dilated in the UVJ obstruction as compared to the UPJ obstruction.
The interstitial inflammation is an important factor contributing to the early damage of nephrons and the renal failure in OU.It constitutes an early response to the unilateral urinary obstruction (3) and this response is mainly mediated by the renal infiltration by macrophages that are attracted by a variety of cytokines and chemokines (4).The inflammation leads to the generation of reactive oxygen species (ROS) (5).The overproduction of ROS has been identified as a key component of apoptotic pathways involving the activation of endogenous endonucleases and the direct DNA fragmentation (6).
Caspases are crucial mediators of programmed cell death (apoptosis).Among them, caspase-3 is a frequently activated death protease, catalyzing the specific cleavage of many key cellular proteins.Caspase-9 is an important initiator caspase, and upon an apoptotic stimulation, caspase-9 further processes other caspase members including caspase-3 to initiate a cascade, which leads to apoptosis (7).Apoptosis has a significant role in the pathogenesis of the renal cellular injury derived from urinary tract obstruction, and the renal tubular cell damage caused by increased hydrostatic pressure is one of the factors regulating the renal apoptotic response.It can provide a powerful mechanical stimulus to apoptosis in the obstructed kidney (8,9).A probable pressure reducer can be the collecting duct itself.The expandable structure of the whole collecting system, including the pelvicalyceal system and the ureter, can balance this pressure overload on the tubular cells.
In this study, our aim was to investigate the differences of apoptotic and inflammatory activity in kidneys with a ureteropelvic or ureterovesical junction obstruction by creating two different obstructive uropathy models in rabbits.

MATERIAL AND METHOD
Twenty-four rabbits were separated into four groups.A UPJ obstruction model was created in the first and the second groups, and a UVJ obstruction model was created in the third and the fourth groups initially.Thirty days after the initial operations, bilateral nephrectomies were performed in the first and the third groups.In the second and the fourth groups, rabbits underwent revision and the obstruction was relieved by surgery at the 30th day.In the second and the fourth groups, bilateral nephrectomies were performed 30 days after the revision.Nephrectomy specimens were evaluated in order to measure the apoptotic activity at protein level, and the pathological specimens were examined by light microscopy following the routine tissue processing.

Surgical Method UPJ obstruction model
A two-cm-long left flank incision was applied to the rabbits.After the medial deflection of the intestinal structures, we advanced to the retroperitoneal space.Kidney was identified and the dissection of the hilum was performed.The modification of the partial ureteral obstruction as defined by Shokeir et al. was used in our model (10).Renal pelvis and ureteral junction were found and suspended.A 0,035-mm guide wire was placed parallel and adjacent to the proximal ureter.A 2/0 silk suture was inserted around the wire and the ureteropelvic junction, and then the suture was tied.The loop point of the suture was lubricated with the lubricating jelly in order not to harm the junction point, and the guide was removed.By this way, ureteropelvic junction size was reduced to the diameter of the wire, and a partial obstruction model was formed (Figure 1a).

UVJ obstruction model
A two-cm-long left inguinal incision was applied to the rabbits.Following the superior deflection of the intestinal structures, we advanced to bladder.The left ureter was found and the dissection of the distal portion of the left ureter was performed.The same partial obstruction model was applied to the UV junction.(Figure 1b).

Revision and obstruction release surgery
Rabbits underwent the same incisions according to their previous surgery, and the same procedures were applied to dissect the junction points.The silk sutures were found in the places which they were applied to 30 days ago.They were cut and removed.A ureteral re-implantation and pyeloplasty were performed by using 7/0 sutures.

Genetic Method Quantitative Real-time PCR
The total RNA isolation from kidney tissue was performed by phenolguanidine thiocyanate extraction using RNAzol RT reagent (Molecular Research Center, Inc. Cincinnati, OH) according to the manufacturer's instructions.Total RNA was reverse-transcribed to cDNA using Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics, Mannheim, Germany) in a 20 µL reaction mixture.Random hexamers were used to prime the cDNA synthesis.A real-time PCR was performed using a Light Cycler 480 II System (Roche Diagnostics, Mannheim, Germany).To quantify cDNA, we performed Real-time quantitative PCR using LightCycler 480 Probe Master mix and hydrolysis probes.Primer and probe used in Real-time PCR for the gene were as follows.

Western Blot Analysis
Tissue samples were homogenized with lysis buffer.Lysates were sonicated and incubated on ice for 5 min before centrifugation at 13,000 g for 15 min at 4 0 C. Supernatants were transferred to microcentrifuge tubes, protein contents were measured using a BCA protein assay kit (PIERCE, Rockford, IL, USA).For electrophoresis, 30 µg of each homogenate was prepared with 4X Laemmli sample buffer and incubated at 95 0 C for 5 min.The samples were loaded into SDS-PAGE gels, and electrophoresis was performed, followed by transfer to PVDF membranes.Nonspecific binding sites were blocked by treating membranes in 5% non-fat dried milk in TBS-Tween 20 for 1 h at the room temperature.To study protein expression, membrane blots were incubated with polyclonal cleaved caspase-3, procaspase-3, procaspase 9, and GAPDH (Stressgen, San Diego, CA) antibody diluted in TBS-Tween 20 0.1% plus non-fat dried milk 5%.Thereafter, membranes were washed three times for 10 min and incubated for 1 h at RT with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology, Beverly, MA, USA).Immuno-detection was performed with SuperSignal West Pico Chemiluminescent Substrate system (PIERCE, Rockford, IL, USA), according to the manufacturer's instructions and then exposed to CL-Xposure film (PIERCE, Rockford, IL, USA).Western blots were quantified using the Image J 1.32 software after densitometric scanning of the films.

Pathological Examination
Nephrectomy specimens from each animal were fixed in 10% buffered formalin for 12 hours and processed for routine paraffin embedding.The 4 µm sections were prepared from paraffin blocks and stained with hematoxylin-eosin, trichrome and periodic acid-Schiff (PAS) stains.The tubulointerstitial injury was examined semi quantitatively in renal cortical parenchyma, with X20 objective and scored on a scale of 0 to 4, as follows: 0, no tubulointerstitial injury, 1, <25% of the tubulointerstitium injured; 2, 25-50% of the tubulointerstitium injured; 3, 51 to 75% of the tubulointerstitium injured; 4, >75% of the tubulointerstitium injured (11).Tubulointerstitial injury was defined as tubular dilation, tubular atrophy, sloughing of tubular epithelial cells and interstitial fibrosis with inflammation.Interstitial fibrosis was examined on Masson's trichrome stained sections.The presence or absence of focal segmental and global glomerulosclerosis in the renal cortical parenchyma was also recorded.Since neither of them was observed in the nephrectomy specimens, scoring was not applied.

Statistics
Expression of the target genes' mRNA relative to that of the housekeeping gene mRNA was calculated using the relative expression software tool (REST 2009, V2.0.13), and statistical significance was determined using the pair-wise fixed reallocation randomization test.P<0.05 was considered statistically significant.Statistical analysis was performed by the Student's t test.

Ethical Committee
This study was approved by Gazi University Ethical Committee with document number GÜET-08.054 and date 17/12/2008.

RESULTS
Normalization of procaspase-9 and procaspase-3 levels according to GAPDH was performed.Relative expression of procaspase-9 and procaspase-3 of the right kidneys were accepted as 1.Procaspase-3 levels were significantly higher in the left operated kidneys than the levels obtained in the right counterparts (Figure 2).When procaspase-9 expression levels were considered, there was no statistically significant difference between the right and the left kidneys.When we gave a statistical score of 1 to the procaspase-9 and cleaved caspase-3 levels in UPJ obstruction, we found that scores for procaspase-9 and cleaved caspase-3 levels were 0.84 and 0.82 respectively (Figure 3).Similarly, scores of procaspase-9 and cleaved caspase-3 levels were found to be 0.86 and 0.84 respectively in the UVJ obstruction.After the revision of UVJ obstruction, scores decreased to 0,67 and 0,6 respectively.To determine the role of inflammation in the pathogenesis of obstructive uropathy, normalization of COX-2 levels according to GAPDH was performed.When the relative expression of COX-2 levels in UPJ obstruction was accepted as 1, revision of obstruction led to the relative expression level of 0.79 for COX-2.In UVJ obstruction, the relative expression of COX-2 level was 0,74.After the revision of the UVJ obstruction, the relative expression of COX-2 level decreased to 0.38 (p<0.05)(Figure 4).The increase in procaspase-9 and cleaved caspase-3 activities in the ureterovesical obstruction group was less than that of the ureteropelvic obstruction group (p<0.05,p<0.01, respectively).
After the revision of obstruction, the decrease in procaspase-9 and cleaved caspase-3 activities was more significant in Group 4 than in Group 2 (p<0.05,p<0.01, respectively).The decrease in COX-2 expression was insignificant in Group 2 and 4 when compared to Group 1 and 3 (p>0.05,p>0.05, respectively).

Histopathologic Findings
Although mean histopathological scores of interstitial fibrosis, interstitial inflammation and tubular atrophy were less severe in the revised group of UPJ obstruction as compared to the unrevised counterpart, only the score for interstitial fibrosis showed a statistically significant difference between the groups (p<0.05).The results for the UVJ obstruction group and its revised counterpart were similar for the pathologies examined and were not statistically significant (p value for interstitial fibrosis: >0.05, p value for interstitial inflammation: >0.05 and p value for tubular atrophy: >0.05).
Mean scores obtained from histopathological analysis were summarized in Table 1. Figure 4 represents the histopathological findings of renal parenchyma in different groups.

DISCUSSION
The urinary obstruction results in hydronephrosis which can cause renal parenchymal damage.The level, severity and duration of obstruction causes varying degrees of obstructive uropathy.The effects of obstructive nephropathy, including apoptosis and cell death, glomerulotubular injury, tubular dilatation, interstitial inflammation, and progressive interstitial fibrosis (12), can be considered as the result of consecutive cellular events.Progressive fibrosis is triggered by interstitial inflammatory cell infiltration (13), myofibroblastic activation and extracellular matrix deposition (14,15).Renal tubular apoptosis is activated by transforming growth factor-β1(TGF-β1) (16), tumor necrosis factor-α (TNF-α) (17), Fas (18), p53 (19) and caspases (17).The down regulation of some growth factors such as epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) facilitates the progression of apoptosis as well (20).
Increased activity of the renin-angiotensin system and other vasoconstrictor systems are the first hemodynamic changes observed in the obstructed kidneys (18).The amount of urine increases in the collecting system, and this leads to the increase in the interstitial pressure.Accordingly, the hydrostatic pressure reflecting on the tubules increases, and the tubular distention damages the tubular epithelial cells ( 21).An interstitial inflammatory response is initialized by a macrophage infiltration afterwards.COX-2 has an essential role in the synthesis of prostaglandins which are associated with the inflammatory process.Norreguard et al. investigated the contribution of COX-2 to ureteric motility and pelvic pressure after obstruction (22).They showed increased COX-2 labeling in surface epithelium and smooth muscle layers of both rat and human proximal dilated ureters as compared to control ureters.They concluded that COX-2 activity contributes to the increased pelvic pressure after obstruction.In our study, the expression level of COX-2 was highest in the UPJ obstruction model.After the revision procedures, COX-2 expression levels decreased insignificantly both in UPJ and UVJ obstruction models.These results show that the inflamatory burden reached in UPJ obstruction is much more as compared to the UVJ obstruction, and their revision provides a reasonable recovery.
There is also a massive myofibroblastic accumulation in the interstitium.These cells lead to the contraction of the kidney parenchyma and the interstitial fibrosis.In our study, interstitial fibrosis increased after partial obstruction at UPJ and UVJ.After revision, histological scores significantly returned to the normal levels.Moreover, reactive oxygen species are significantly increased in the obstructed kidneys, and this is a factor that reduces the threshold of apoptosis (23).One would expect that the results observed in the apoptotic activity would be paralelled by the histopatological data, but the difference was not significant except for the interstitial fibrosis in our study.Perhaps this discrepancy may be the result of a more sensitive response to the increased intraluminal hydrostatic pressure at the molecular level than at the tissue level.
The relief of obstruction carries upmost importance for the recovery of uropathy but the appropriate timing for the treatment is still controversial for different obstruction entities.In this manner, many studies with different obstruction durations have investigated the recovery of the kidney with improved renal functions (24,25).Recently, Soliman et al. (26) in a unilateral ureteral obstruction model for four weeks, showed the recovery of renal functions by ≈ 51% of baseline values at 8 weeks after the relief of ureteral obstruction.In addition, Zhou et al. demonstrated that apoptosis in the renal parenchyma was significantly increased in the the obstructed group as compared to sham group, especially at the 28 th day in an experimental animal model (27).Since apoptotic index was scientifically proved to be increased at the 30 th postoperative day for the revision of the obstruction in animal models, we have also chosen this day for the revision of obstruction in our study.We found significant difference in procaspase-9 and procaspase-3 levels between the control right kidneys and the operated left counterparts.The expression levels in the right kidneys were significantly lower than the left kidneys.We can draw a conclusion that our model of ureteral obstruction at different anatomical localizations was successful.
With regard to the idea of the harmful effect of increasing pressure in the collecting system, the volume of the affected collecting duct may determine the progression of tubuloepithelial damage.Mechanical forces from hydronephrotic distension are transmitted to the renal tubular cells (28), and the mechanical stretch related to the increase of intratubular pressure might in part mediate obstruction-induced apoptosis (29).The mechanism of the stretch-induced apoptosis has been studied in renal epithelial cells by Nguyen et al (30).In their model, certain cell lines of various components in nephron underwent cyclic stretch and apoptotic index of these cell lines was determined (30).Stretched cells displayed an increased activity of caspase-3 and -9, and apoptosis was modulated by the inhibition of caspase-3 and to a lesser extent by caspase-9.They concluded that the stretch induced apoptosis in renal epithelial cells is dependent on the activation of caspase-3 and 9.In our study, similar to Nguyen et al, we found that procaspase-3 expression levels increased but procaspase-9 levels were not altered after the obstruction of UPJ and UVJ in left kidneys when compared to the normal right counterparts.
In addition, this increased apoptotic activity was more prominent in the UPJ as compared to the UVJ.In this respect, ureterovesical obstruction may provide more volume in terms of decreasing the pressure reflecting on the tubular cells with the significant contribution of ureter volume to the collecting duct.Therefore, ureter may be acting like the guarding angel of the nephron in this case.

Ureter guardian angel 13
Because of its severe expandability, ureter can reach significant volumes.Due to the relieving effect, less pressure might be reflected to the renal parenchyma, and this might lead to the less stretched nephrons.
Moreover, Nguyen et al. found that a 5-25% change in the maximal radial stretch did result in an increase in apoptosis, but the frequency of stretch did not affect the outcome (30).This indicates that the stretchinduced apoptosis is dependent upon maximal radial change rather than the frequency of cyclic stretch.Our study results were consistent in that rabbit kidneys with a UVJ obstruction had rather more benign expression levels of apoptotic proteins than those with a UPJ obstruction.Unfortunately, our study was limited in terms of interpreting the volume changes in each group.It would be more illustrative, if data relative to the pyelocalyceal pressures and diameters could also be pre-and post-operatively evaluated and presented, endorsing our conclusions.
The results of our study showed that the relief of obstruction gives a hand for better improvement for renal function after obstructive uropathy.Apoptotic expression levels were recorded at the lowest level in the UVJ revision group.This is further logical in that the group of UVJ obstruction suffered less than the group of UPJ before the revision.And especially in low volume obstructions like UPJ obstruction due to the low compensation capacity of the renal pelvis, we could reach better molecular results in the UVJ group.However, we approach our results critically in that we did not perform a radioisotope renography following the revision surgeries to determine whether we have achieved a non-obstructive curve in our study.This could be a subject of further studies in this issue.

CONCLUSION
The rabbits with a UVJ obstruction had lower apoptotic indexes as compared with those with a UPJ obstruction.The apoptotic activity decreases to almost normal levels after adequate revision of obstruction in both groups, but this improvement is more significant in the UVJ obstruction.

Figure 1 :
Figure 1: Macroscopic view of; a) Partial ureteropelvic obstruction model b) Partial ureterovesical obstruction model c) The right and left kidneys after sacrifice.

Figure 4 :
Figure 4: a.Normal cortical parenchyma of a right rabbit kidney, without any inflammation or fibrosis and tubular atrophy, H&E X40. b.Minute cortical scar characterized by focal fibrosis with atrophic tubules and accompanying mild inflammatory infiltrate, in the kidney of a rabbit with ureterovesical obstruction, group 3, H&E X 40.c.Moderately large segmental scarring area of the rabbit kidney, contrasting with the surrounding normal looking cortical parenchyma, group 1, H&E X40.

Table 1 .
Mean histopathologic scores of each groups.