Glomerular cell proliferation and apoptosis in uninephrectomized spontaneously hypertensive rats

Kidney International, Vol. 54, Suppl. 68 (1998), pp. S-36 –S-40

MECHANISMS OF HYPERTENSIVE RENAL DAMAGE

Glomerular cell proliferation and apoptosis in uninephrectomized spontaneously hypertensive rats ANA M. RODR´IGUEZ-L´ OPEZ, OLGA FLORES, MIGUEL A. AR´ EVALO, JOS´ E M. L´ OPEZ-NOVOA Instituto Reina Sofı´a de Investigacio ´n Nefrolo ´gica, Departamento de Fisiologı´a y Farmacologı´a, and Departamento de Anatomı´a e Histologı´a, Universidad de Salamanca, Salamanca, Spain

Glomerular cell proliferation and apoptosis in uninephrectomized spontaneously hypertensive rats. We studied renal function, glomerular cell proliferation and apoptosis for three months after uninephrectomy (UNX) in young, male, spontaneously hypertensive rats (SHR). Apoptosis was assessed by in situ dUTP biotin nick-end labeling method (TUNEL) and by propidium iodide staining. Proliferation rate was determined by immunohistochemistry to proliferating cell nuclear antigen (PCNA). Glomerular bcl-2 expression was assessed by Northern blot analysis. Our results indicate a parallel increase in proliferation and in apoptotic rates in glomerular cells from the first to the second month after UNX. In the third month after UNX, PCNA-labeled cell number continues increasing, whereas TUNEL-labeled cells did not increase. Bcl-2 expression was negative in the first and second months and increased in the third month. Glomerular size and proteinuria increased progressively along the three months of follow-up. Our observations demonstrate a different profile of cell proliferation and apoptosis during the genesis of early glomerular damage in UNX- SHR.

In addition to intraglomerular hypertension, glomerular enlargement is also closely related to the development of scarring [1]. Several authors have reported that glomerular injury associated with hypertrophy may be dissociated from increases in glomerular pressure [1– 4]. The injurious effects of increased glomerular size have also been ascribed to a heightened capillary wall tension [4]. Laplace’s law indicates that, for a given intravascular pressure, the wall tension is increased with the diameter of the vessel. Thus, the hypertrophied, enlarged glomerular capillary is more sensitive to the damaging effect of any increment in intraglomerular pressure [4]. A link between mesangial cell proliferation and subsequent glomerular sclerosis has been now established in various forms of renal injury including glomerulonephritis, glomerular hypertension and diabetes [4]. Glomerular size and cell number is also regulated by programmed cell death or apoptosis [5]. Apoptotic cell

Key words:: renal fibrosis, hypertension, SHR rats, apoptosis, PCNA, TUNEL.

© 1998 by the International Society of Nephrology

death has been observed in glomeruli from humans with proliferative types of glomerulonephritis [6], and in mesangial cells of anti-Thy 1 antibody-induced glomerulonephritis [7]. Recently, an increased degree of apoptosis has been reported in the rat remnant kidney model [8]. However, this model is characterized by massive renal mass ablation, and the apoptotic phenomenon could be related to the accelerated renal hypertrophy instead of glomerular sclerosis. By contrast, the SHR represents a model of severe systemic hypertension without a reduction of renal function. These animals develop a slowly progressive renal damage with moderate proteinuria and glomerular sclerosis when the rats become aged [9]. The renal disease is more severe and develops more rapidly if SHR undergo unilateral nephrectomy (UNX) [10]. However, even after UNX, the progression of renal disease is slow. Furthermore, and in contrast with other models of hypertension associated to radical renal mass reduction, the 50% reduction in glomerular filtration rate (GFR) imposed by UNX is similar in magnitude to the degree of functional impairment initially observed in many patients at the risk of progressive renal failure. Thus, the UNX-SHR seems to represent a good experimental model to evaluate the mechanisms that lead to glomerular and tubulointerstitial fibrosis associated to hypertension and renal failure. Hamet et al have reported increased apoptosis in the heart from spontaneously hypertensive rats (SHR) and in the heart, kidney (inner cortex and medulla) and brain from spontaneously hypertensive mice [11]. In the present study, we assessed glomerular cell apoptosis and proliferation in the first three months after UNX in spontaneously hypertensive rats. METHODS Experimental model Seven weeks, male SHRs were obtained from Charles River (France), and acclimated to the environment for one week. Then, animals underwent a unilateral (left) nephrectomy under ether anesthesia in aseptic conditions. After recovering from anesthesia, rats were housed in standard

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cages in a daylight (12 hr), temperature (20°C) and humidity (60%) controlled room. Rats were allowed to free access to food (standard chow containing 20% protein by weight) and water. Every month after unilateral nephrectomy, rats were placed into metabolic cages, with free access to food and water during four equilibration days and two more days to collect samples of blood and urine to determinate proteinuria and creatinine clearance. Endogenous creatinine clearance was used to estimate glomerular filtration rate (GFR). Urinary protein excretion was determined by the method of Bradford. Systolic and diastolic arterial pressure and heart rate were measured every month in awake animals with a tail cuff method (Electrosphignomanometer, LE 5100; Letica, Barcelona, Spain). One, two and three months after unilateral nephrectomy, five animals randomly chosen were anesthetized with ether and a renal perfusion with saline through the abdominal aorta was performed to wash the blood out from the kidneys. Kidney tissue was used for histochemical and immunohistochemical studies. For morphometric studies, 5 mm thick sections were stained with syrium red and the glomerular area was determined by computer-assisted image analysis [12, 13]. DNA fragmentation associated with apoptosis was detected in situ by the TdT-mediated dUTP-biotin nick-end labeling (TUNEL) method [14]. To detect the proliferative cell nuclear antigen (PCNA) an immunohistochemical staining using a mouse monoclonal antibody to PCNA (dilution 1:50) (Cormedica Corp., CA, USA) was performed. Omitting the specific antibody in the first antibody incubation step assessed the specificity of the immunostaining. Glomerular bcl-2 (B-cell lymphoma-leukemia-2 protooncogene) expression was assessed by Northern blot analysis. Glomeruli were isolated from the kidneys by mechanical sieving [15]. Total cellular RNA was isolated from rat glomerular homogenate using the guanidinium thiocyanate-phenol-chloroform method [16]. The cDNA probes used were murine bcl-2 (provided by Dr. A. Ortiz, Fundacio ´n Jime´nez Dı´az, Madrid, Spain) and b-actin (ATCC), which was used as an internal control probe. Statistical methods The Kolmogorov-Smirnov test was used to assess normality of the data distribution. One way analysis of variances and Scheffe´’s test were used for normally distributed data. Kruskal-Wallis multiple comparison Z-value test was used for non-Gaussian data. A P value lower than 0.05 was considered statistically significant. For the quantification of the TUNEL method and PCNA immunohistochemistry, at least 30 glomerular sections were examined for each animal and the number of positively stained nuclei per glomerulus was determined. An average of these values for each rat were considered as the TUNEL and PCNA indices, respectively.

Table 1. Hemodynamic and renal function parameters First month (N 5 15) Mean arterial pressure 204.5 6 5.2 mm Hg Heart rate bpm 384.4 6 7.1 Creatinine clearance 0.74 6 0.085 ml/min Urinary protein excretion 12.36 6 1.03 mg/day

Second month Third month (N 5 10) (N 5 5) 208.0 6 4.5

224.7 6 5.4a

370.5 6 8.1 0.64 6 0.09

379.1 6 10.1 0.48 6 0.1a

19.70 6 2.24a

24.40 6 4.13a

Values are mean 6 SEM. a P , 0.05 vs. first month

RESULTS Mean arterial pressure was significantly increased along the experimental period, while heart rate did not change significantly with time (Table 1). Creatinine clearance decreased in the third month after nephrectomy. Urinary protein excretion was progressively increasing from the first to the third month of treatment (Table 1). Glomerular section area increased progressively from the first to the third month after nephrectomy. Medians are in the first month 10629 mm2, in the second 12731 mm2 (P , 0.05 vs. first month) and 18436 mm2 in the third month (P , 0.05 vs. either first and second month). Figure 1A shows a representative image of a glomerulus with some apoptotic cells stained by the TUNEL method to detect apoptosis. One month after nephrectomy a minimal number of glomerular cells were stained. The number of apoptotic cells increased significantly in glomeruli at the second month, with a slight, nonsignificant decrease at the third month (Fig. 2A). The TUNEL technique stained nearly all the cells in the positive control sections treated with DNAase before the Tdt reaction (data not shown). Figure 1B shows a representative image of a glomerulus from an uninephrectomized SHR after immunohistochemical staining for PCNA. Glomerular PCNA score was significantly increased at the second month of the study. A further increase was observed at the third month (Fig. 2B). Glomerular expression of bcl-2 mRNA was detected by Northern blot (Fig. 3) at the third month after nephrectomy, whereas it was undetectable during the first and second months, b-actin-mRNA expression, used as housekeeping gene, did not change with time after UNX. DISCUSSION The present study reveals that unilateral nephrectomy in 8-week-old, male SHR induces a progressive renal damage characterized by proteinuria and an increase in corpuscular section area. Glomerular changes can be observed as soon as one month after UNX, confirming that kidney removal accelerates renal damage in this model of genetic hypertension [10].

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Fig. 1. Representative microphotographs showing: (A) TUNEL positive (brown) nuclei in glomeruli and (B) PCNA expression (red) in glomeruli from uninephrectomized SHR (3510).

The progression of glomerular damage is often associated with mesangial changes such as mesangial proliferation and mesangiolysis [17, 18]. The hypercellularity resulting from cell proliferation seems to play a major role in the

genesis of glomerular damage in several experimental models [17]. We have observed a progressive increase in the number of glomerular proliferating cells, assessed by immunohistochemistry for PCNA, which is accompanied by

Rodrı´guez-Lo ´pez et al: Hypertension and renal apoptosis

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Fig. 3. Northern blot for bcl-2 and b-actin. RNA was obtained from glomeruli from uninephrectomized SHR, one month (lane 1), two months (lane 2) and three months (lane 3) after nephrectomy.

Fig. 2. Semiquantitative evaluation of (A) TUNEL positive nuclei and (B) PCNA positive nuclei in glomeruli from SHR, one, two and three months after nephrectomy. Asterisk represents significant difference (P , 0.05) versus the first month.

a time-dependent increase in the glomerular cross-sectional area. After uninephrectomy, an increased expression of platelet-derived growth factor (PDGF) and other growth factors have been reported in SHR, due to an increase in intraglomerular pressure [19]. This fact could lead to glomerular cell proliferation and the subsequent enlargement of glomerular size. The whole number of glomerular results from the balance between cell proliferation and cell death. Recently, an increased degree of apoptosis has been reported in the rat remnant kidney model [8]. However, this model is characterized by massive renal mass ablation and the apoptotic phenomenon could be related with the accelerated renal hypertrophy instead with hypertension and glomerular sclerosis. In the present study, we have observed an increase in the number of glomerular apoptotic cells in the

second month after UNX, with respect to the first month. However, in the third month after nephrectomy, apoptotic rate was reduced. In this last stage a probable failure in the clearance of renal cells by apoptosis, together with a sustained proliferative activity lead to the increased glomerular size. It should be taken into account that apoptotic cells are scarce, even in a tissue in which a great number of cells are being removed, due to the rapid clearance of apoptotic phenomenon [20], thus explaining the low number of cells labeled by TUNEL method within the glomeruli. The protein Bcl-2 is a negative regulator of apoptosis that can block apoptotic processes and extends the lifespan of lymphoid cells [21]. The expression of bcl-2 is not specific to neoplasic cells and it has been also identified in a number of non-hematolymphoid tissues [22]. In the present study we observed that in the third month after UNX, bcl-2 expression was increased, coinciding with the lack of increase in glomerular cells apoptotic rate. Thus, it can be suggested that this bcl-2 expression could play a role in the slight reduction in apoptotic rate. In conclusion, the present study in uninephrectomized SHR demonstrates a time dependent increased glomerular growth and glomerular cell proliferation. Glomerular cell apoptosis increased from the first to the second month after nephrectomy, but decreased in the third month. Our results suggest that an imbalance between cell proliferation and apoptosis could play an important role in the genesis of

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glomerular hypertrophy associated to hypertension. The expression of bcl-2 in the third month after unilateral nephrectomy could explain this change in the pattern of apoptosis observed.

10. 11.

ACKNOWLEDGMENTS We thank to Ms. A. Pe´rez (Depart. Anatomı´a Patologica, Hospital Clı´nico, Salamanca, Spain) for technical assistance in the histochemical and immunohystochemical studies. This study has been supported by a grant from the Comisio ´n Interministerial de Ciencia y Tecnologı´a (CICYT grant SAFT-95-0533). Reprint requests to Prof. J.M. Lo ´pez-Novoa, Departamento de Fisiologı´a y Farmacologı´a, Facultad de Medicina, Universidad de Salamanca, Edificio Departamental, Avenida Campo Charro s/n., 37007 Salamanca, Spain. E-mail: [email protected]

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