EXPERIMENTAL INVESTIGATIONS Cell proliferation and apoptosis in Wistar rat kidney after renal mass ablation and low-dose irradiation

Summary. Cell proliferation and apoptosis in the remnant rat kidney after treatment with low-dose irradiation was investigated. Material and methods. In the first group (n=9), adult male Wistar rats underwent 5/6 nephrectomy (NPX); in the second group (n=9), NPX was combined with low-dose irradiation. Rats without surgery and irradiation formed the control group (n=9). Results. Hypertension and proteinuria induced by NPX were decreased by 3-Gy irradiation. The 5/6 NPX rats showed a dramatic increase in proliferating and apoptotic cells in the glomeruli and in the distal tubules at week 2, which was significantly decreased by low-dose irradiation. Conclusion. The data demonstrate that low-dose irradiation is a factor slowing the process of chronic renal injury.


Introduction
During the last years, the understanding of pathophysiological processes leading to the progression of chronic renal disease has signifi cantly changed and advanced (1). Irrespective of the renal primary detractive mechanism, immune or nonimmune, the remaining parts of the kidney undergo adaptive changes. A critical amount of nephrons are destroyed during the development of chronic renal disease (2), but the remaining ones undergo compensatory changes. Many experiments demonstrate that alterations in glomerular hemodynamics associated with renal ablation are accompanied by structural lesions and changed single nephron hyperfi ltration (1, 2). Five-sixths nephrectomy (5/6 NPX) is a very useful and widely studied model for the analysis of progression of chronic renal disease (3,4). Experimental studies incriminate glomerular hypertension in mediating progressive renal damage after any of a variety of initiating injuries (5, 6). The main mechanisms involved in this process can be different: hemodynamic (7), cellular (1), or molecular (7). Alterations appearing as a result of 5/6 nephrectomy in the remnant kidney give a possibility to study various aspects of the damage (4). Nowadays, knowing the pathogenesis of glomerulosclerosis (8), it is theoretically possible to slow down the process of fi brinogenesis (9), but it is complicated as many factors participate in the process: various interleukins in early stages (9), chemokines (10), cytokines, and growth factors (11). Later structural changes appear in tissues, which will lead to pathological changes in physiological functions of the organ (12). Few effective possibilities exist to mimic early alterations in the kidney with the exception of compounds blocking the renin-angiotensin system (13). It is known from literature that as a result of radiation in therapeutic doses, radiation nephropathy develops in the kidneys (14, 15), but the use of lowdose radiation to slow down the progression of renal disease has been studied insuffi ciently (16). In our previous studies (17), we investigated the effect of low-dose irradiation on the development of chronic kidney disease, and based on our results, it had a renoprotective effect on the kidneys. Chronic renal disease is characterized by perishing of the cells in renal tissue. This process is a particular type of cell death, which has several distinguishing features from necrosis, and is often referred to as programmed cell death or apoptosis (18). Typical patomorphological changes are observed during this process in the tubular epithelial and mesangial cells (8). Apoptosis plays an important role in the regulation of the number of renal cells in the diseased kidneys (19). When the structure of glomerular basement membrane (GBM) is altered, GBM is not able to prevent the passage of proteins with a large molecular mass and diameter, which leads to proteinuria.
Assuming that low-dose irradiation diminishes apoptotic changes in the early stages of chronic kid-ney disease, we investigated the expression of proliferative and apoptotic cells in the remnant kidney after experimental renal 5/6 nephrectomy.

Material and methods
Experimental design and animals. Adult male Wistar rats (Laboratory Animal Center, University of Kuopio, Finland) were studied. An acclimatization period of 10 days was allowed before any experimental work was undertaken. Rats were kept in a climate-controlled facility at the Faculty of Medicine, University of Tartu, where animals were housed under standard conditions on a 12-h light/ dark cycle and fed with standard rodent chow (70, Lactamin AB, Sweden) and tap water ad libitum. At approximately 8 weeks of age, rats weighing 240-262 g were anesthetized with intraperitoneal methohexital sodium (5 mg per 100 g body weight [BW], Brietal, LILLY GmbH, Germany). Renal ablation was then accomplished by right nephrectomy and selective ligation of extrarenal branches of the left renal artery. All together 18 rats were randomized after the surgery, divided into two groups, and studied during two weeks: 5/6 NPX-2wk (n=9), 5/6 NPX + 3 Gy-2wk (n=9). The left kidney of 3 Gy group rats was irradiated ( 60 Cobalt) 24 hours after surgery in anesthetized (Brietal) animals with 3 Gy in a single dose in the Clinic of Radiology, Tartu University Hospital. Healthy Wistar rats (n=9, without surgery) were studied as controls. All animal procedures were approved by the Animal Care Committee, University of Tartu, in accordance with the European Communities Directive of November 24, 1986 (86/609/EEC).
Analysis of kidney function. Systolic blood pressure (SBP, mm Hg) was measured weekly by the tail-cuff method (Harvard Apparatus, USA) in awake, prewarmed rats at weeks 1 and 2. Animals were placed in metabolic cages for 24 h for urine collection, and urine protein and serum creatinine were measured with a Hitachi 912 analyzer. Animals were killed at week 2, and the kidneys were harvested for analysis of sclerosis, apoptosis and immunohistochemical studies.

Morphological analyses.
Kidney tissue was fi xed in 4% formalin (for light microscopy) or 4% paraformaldehyde solution (for detection of apoptosis and immunohistochemistry) and routinely processed. Fourµm sections were stained with hematoxylin-eosin and periodic acid-Schiff. A semiquantitative score was used to evaluate the degree of focal segmental glomerulosclerosis (FSGS). Sclerosis was defi ned as a collapse of glomerular capillary tuft accompanied by hyaline material. The severity of sclerosis for each glomerulus was graded for the presence of FSGS ac-cording to the following scale: 0, no evidence of focal segmental glomerulosclerosis; I, <25% involvement; II, 25-50% involvement; III, >50% involvement of the glomerulus, respectively. Two independent observers in a blinded fashion performed the evaluations. An Olympus BX-50 microscope was used for viewing and photographing.
Immunohistochemistry. Proliferating cells were identifi ed by the expression of proliferating cell nuclear antigen (PCNA). Three-μm cryostat sections were incubated with the purifi ed hamster antimouse monoclonal antibodies to PCNA (BD, PharMingen, USA), diluted in phosphate-buffered saline pH 7.4 (PBS) 1:50, for 30 min at room temperature. Slices were washed in PBS (3 times) and incubated with FITC-conjugated antihamster IgG monoclonal antibodies (diluted 1:50, BD, PharMingen, USA) for 30 min at room temperature. Negative controls with nonspecifi c antisera instead of primary antibody were done at the same time and showed no staining. For immunofl uorescence, sections were examined at a magnifi cation of ×100, ×368 with a Zeiss Axiophot 2 microscope. The results were evaluated according to the following scale: 0, none; 1+, isolated staining in less than 25% of the glomeruli; 2+, staining in 25 to 50% of the glomeruli; 3+, staining in 50 to 75% of the glomeruli; 4+, staining present in more than 75% of the glomeruli. Two independent observers in a blinded fashion performed the evaluations.

Detection of apoptosis.
Apoptotic cells were detected by the transferase-mediated dUTP nick-end labeling (TUNEL) method using in situ cell death detection kit (POD Cat No. 1684817 Roche Diagnostics, Roche, Germany). Briefl y, 4-μm paraformaldehyde-fi xed sections were deparaffi nized and rehydrated in graded ethanol and PBS. Samples were pretreated by incubation with proteinase K (2 μg/mL; Roche Diagnostics GmbH, Mannheim, Germany) for 15 min at room temperature. Endogenous peroxidase was inactivated by 3% H 2 O 2 (Merck, Germany) in PBS for 30 min, and sections were rinsed with PBS, immersed in citrate buffer pH 6.0, and then incubated with TdT and digoxigenin dUTP (diluted 1:1) at 37°C for 60 min. Then the reaction was stopped with buffer, and antidigoxigenin peroxidase conjugate was applied and incubated for 30 min. The slides were developed by using diaminobenzidine substrate. For negative control, slides were incubated with TdT buffer without TdT. As a positive control, slides were treated with DNase (1 μg/mL; Sigma, St. Louis, USA) for 10 min. Cell apoptosis in the cortex of the kidney was assessed by scoring the TUNEL-positive cells in the glomeruli at ×100 magnifi cation in all glomeruli in each section. TUNEL-positive staining was graded from 0 to 4: 0, none; 1+, isolated staining in less than 25% of the TUNEL-positive cells; 2+, staining in 25 to 50% of the TUNEL-positive cells; 3+, staining in 50 to 75% of the TUNEL-positive cells; 4+, staining present in more than 75% TUNEL-positive cells. Two independent observers in a blinded fashion performed the evaluations. Sections were examined at a magnifi cation of ×100, ×368 with a Zeiss Axiophot 2 microscope.
Statistical analysis. Data are presented as mean values ± SEM. Data were analyzed by one-way ANOVA.

SBP and renal function.
At week 1, SBP was increased in 5/6 NPX + 3 Gy group rats, but not in rats of the 5/6 NPX group. At week 2, SBP was clearly increased in the 5/6 NPX group, while in the 5/6 NPX + 3 Gy group, SBP increase was lower (Table 1).
To assess renal function, the levels of proteinuria and serum creatinine were determined. Twentyfour-hour urinary protein excretion was increased in both the operated groups. Maximal effects on proteinuria were seen in the 5/6 NPX group animals, but rats irradiated with 3 Gy showed lower levels of proteinuria (Table 1). Serum creatinine at week 2 was signifi cantly higher in the 5/6 NPX group as compared to the 5/6 NPX + 3 Gy group (Table 1).

Morphological analyses.
Morphological studies demonstrate that glomerular changes were associated with renal mass ablation. The FSGS increased signifi cantly in the 5/6 NPX group (Table 2; Fig. A) as compared to the control group. In the 5/6 NPX + 3 Gy group, FSGS was decreased as compared to the 5/6 NPX group (Table 2; Fig. B).
Immunohistochemistry. The 5/6 nephrectomized rats showed a dramatic increase of PCNA-positive cells in glomeruli at week 2 ( Table 2; Fig. C). The mean number of positive cells per glomerular cross section was determined by evaluating 15-20 glomeruli. PCNA positivity was present in the glomerular epithelial cells, mesangial area, and also in tubules (Table 2). In contrast, all irradiated rats showed signifi cantly fewer PCNA-positive cells at week 2 than the 5/6 NPX group (Table 2; Fig. D). Glomerular cell proliferation was negative in the glomeruli and distal tubules of healthy rats (Table 2). Values are given as mean±SEM. Statistically signifi cant diff erences (at P≤0.05) are shown as folows: a 5/6 NPX group vs. 5/6 NPX + 3 Gy group; b 5/6 NPX vs. control; c 5/6 NPX + 3 Gy group vs. control. ANOVA was used for intergroup comparison among groups (Tukey and Dunnett tests).

Apoptosis. Apoptotic cells were detected in the glomerular and epithelial cells of the remnant kidneys by the TUNEL method. The number of apoptotic cells was signifi cantly increased in the 5/6
Control, healthy animals (rats without surgery and irradiation); 5/6 NPX, nephrectomized rats; 5/6 NPX + 3 G, nephrectomized rats with irradiation. Values are given as mean±SEM. Statistically signifi cant diff erences (at P≤0.05) are shown as follows: a 5/6 NPX group vs. 5/6 NPX + 3 Gy group. ANOVA was used for intergroup comparison among groups (Tukey and Dunnett tests).

Discussion
We have previously shown that low-dose irradiation has a benefi cial effect on the course of experimental renal disease employing the renal mass ablation model. Irradiation of 3 Gy in the 5/6 nephrectomized rats diminished proteinuria and hypertension as well decreased serum creatinine, FSGS, and PCNA at week 2 (17). Having reproduced these fi ndings in the current study (see Table 1 and 2), we aimed to investigate how the effect of low-dose irradiation is related to the number of apoptotic cells in the glomeruli and tubules after renal mass ablation. Apoptosis has been proposed to play an important role in the regulation of renal cell number in both healthy and diseased kidneys (19). An effi cient deletion of excessive, damaged, or nonfunctioning renal cells and infi ltrating infl ammatory cells by apoptosis seems to be benefi cial (19). During the progression of experimental glomerulonephritis to end-stage renal failure, apoptosis also seems to play an essential role in the resolution of intra-and extraglomerular infl ammation (20). However, the loss of resident renal cells by uncontrolled apoptosis is detrimental as it may induce a reduction of functional renal mass and lead to renal insuffi ciency. Thomas et al. (21) have reported a progressive and sustained increase in the number of apoptotic cells in the glomeruli, tubules, and interstitium of the remnant rat kidneys, with maximal areas of apoptosis detected in the sclerotic glomeruli, atrophied tubules, and expanded interstitium, which indicate that apoptosis of renal cells might contribute to the progression of tubular atrophy and chronic renal fi brosis.
Our study and studies by other authors (22) have shown that low-dose irradiation of the remnant kidneys provokes a benefi cial change in the course of renal disease. Many factors may contribute to this favorable feature including lower levels of systemic arterial pressure, as hypertension can be one of the causes of chronic renal insuffi ciency. As a result of hypertension, the different segments of the nephron are damaged fi rst due to the high hydraulic pressure and sclerosis of the glomeruli. In the case of chronic renal insuffi ciency, renal tissue and tubules are destroyed, and because of that, the speed of glomerular fi ltration decreases. Concur-rent to the destruction of the tubules, compensatory changes also occur. Undamaged tubules hypertrophy in order to retain a possibly high speed of glomerular fi ltration. High blood pressure, fi rst of all, damages the glomeruli and leads to the development of glomerular sclerosis. Glomeruli become hypertrophied; mesangial cells show a tendency to proliferation, and their number and size increase. Therefore, it is not surprising that tight blood pressure control can decrease apoptosis of tubular and glomerular cells during renal damage. Soto et al. have reported that renal mass reduction and hypertension caused severe renal lesions associated to an increment of apoptosis rate in glomerular and especially in tubular cells, but tight blood pressure control decreased the apoptosis rate and morphologic lesions (23). Furthermore, the authors suggest that changes in the expression of apoptosis-regulatory genes contribute to the progressive damage in hypertensive rats with renal mass reduction. Decreased apoptosis of tubular and glomerular epithelial cells found in the 5/6 NPX+3 Gy group in our study ( Table 2) thus represents one of the mechanisms of benefi cial actions of low-dose irradiation.
Another benefi cial effect of low-dose irradiation on the course of renal disease in our study was a reduction of proteinuria. Proteinuria is not only a sign of renal insuffi ciency but it is also important in the development of chronic renal insuffi ciency (17). Epithelial cells of nephrons are damaged because of excessive protein content in urine (20). Our previous experiments (24) showed that a modest thickening of the glomerular basement membrane after the 5/6 NPX and changes in the fi ltration pores made the penetration of the barrier possible also for proteins with a large molecular mass. Another factor contributing to the permeation of proteins was hemodynamic, because with the slowing of blood circulation after the ligation of blood vessels, factors contributing to the permeation of proteins appeared. Probably the changes in the glomerular basement membrane are affected by the developing proteinuria, which is accompanied by changes in the fi ltration of proteins. In our experiment, changes in kidney function were expressed by a signifi cant increase in systolic blood pressure in nephrectomized animals by the end of week 2. The NPX group animals had a signifi cantly increased proteinuria and an increased amount of serum creatinine in urine (Table 1), which are indicators of the above-mentioned damages in the fi ltration barrier.
Recently, in connection with the introduction of new immunological, molecular biological, and histological methods, the understanding of the mechanism of progression of renal damage has significantly improved (1,8). In the progression of chronic kidney disease (5), an important role is played by hemodynamic as well as nonhemodynamic factors. In the case of chronic renal failure, as mentioned above, cell proliferation and apoptosis develop in the damaged glomeruli and tubules, in particular the distal ones. With the progression of FSGS, the sclerotic process involves occasional capillaries of the glomeruli, and at the same time, regular proliferation of mesangial cells is noted. Due to the hypertrophy of the glomeruli, also mesangial cells, which show a tendency of proliferation, are damaged. Glomerulosclerosis is characterized by increased extracellular matrix formation and cell proliferation (25). Treatment of nephrectomized animals with irradiation signifi cantly lowered the FSGS, and therefore it is speculated about a possible infl uence of resi-dent mesangial cells on the early events following renal mass ablation and maintenance of subsequent pathophysiological changes (25).

Conclusions
The presented results demonstrate that low-dose irradiation is a factor slowing the process of chronic renal injury. We also found that glomerular cell proliferation and programmed cell death were decreased after treatment with low-dose radiation.