ULTRASTRUCTURAL EVALUATION OF RENAL CONVOLUTED TUBULES OF FEMALE RATS, WHICH HAD PREGNANCY COMPLICATED WITH SECONDARY PREECLAMPSIA

Agnieszka Pedrycz¹, Marcin Wieczorski¹, Zbigniew Boratyński², Krystyna Czerny³, Stanisław Flieger^2
1 Department of Histology and Embryology, with Lab of Experimental Cytology, Medical University of Lublin, Poland
² Deaprtment of Animal Anatomy and Histology, Agricultural University of Lublin, Poland
³ Department of Histology and Embriology, with Lab of Experimental Cytology, Medical University of Lublin, Poland

ABSTRACT

The purpose of present study was analysis of structure of convoluted tubules epithelial cells, which were coming from female rats, which had pregnancy complicated with preeclampsia after nephrotic syndrome induced by adriamycin. In experiment were used 16 female rats divided into two groups: experimental and control (each group contain 8 individuals). Rats from experimental group had administered intraperitoneally adriamycin in dose 5 mg/kg of body weight to induce nephrotic syndrome. Female rats were paired with males 4 weeks from drug administration, and decapitated 4 weeks after delivery. Systolic blood pressure was taken from animals on 10th, 20th day of pregnancy and 10 days and 4 weeks after delivery. In urine from 24 hour collection on 19^th day of pregnancy and 27 days after delivery protein concentration was determined. Histological sections from collected kidneys were observed in electron microscopy. Results show the significant damage in epithelial cells in convoluted tubules with features of the apoptosis. Presence of homogenous casts in tubular lumen was the evidence of excessive proteinuria. The histological changes were similar to that which appear in convoluted tubules rat females during pregnancy complicated with preeclampsia. However features of preeclampsia (hypertension, proteinuria, edemas) present during pregnancy disappeared or decreased starting from 10th day after delivery.

Key words: St post preeclampsia, tubules of kidneys, rat?s mothers, electron microscopy, adriamycin.

INTRODUCTION

Model of secondary preeclampsia, which is the result of nephrotic syndrome existing in female before planned pregnancy is possible to reach on rats. In typical preeclampsia, hypertension appears in the middle of pregnancy, about 10^th day, and edemas and proteinuria present in nephrotic syndrome are increasing during pregnancy. [1, 4, 11, 12] That preeclampsia is induced by administration of adriamycin in single dose 5 mg/kg of body weight 4 weeks before planned pregnancy. [7]. Nephrotic syndrome which appears after 4 weeks [6] during pregnancy had features of secondary preeclampsia (with the background of nephrotic syndrome). Preeclampsia or gestosis appears only in Primates. In lab animals only secondary preeclampsia with the background existed before kidney disease could be obtained. [9].

The purpose of this study was the analysis of the structure of epithelial cells building convoluted tubular wall, in female rats, which had pregnancy complicated with preeclampsia after nephrotic syndrome induced by adriamycin. The features of preeclampsia (hypertension, proteinuria, oedemas) were also evaluated during pregnancy and after puerperium.

MATERIALS AND METHODS

The experiment was performed on 16 female rats with initial body weight 200-250g aged from 2,5 to 3 months. The animals were divided into two groups (experimental and control, 8 rats in each group). The animals from experimental group had administered adriamycin in dose 5 mg/kg of body weight intraperitoneally, to induce nephrotic syndrome. The female rats were paired with males 4 weeks after adriamycin administration, and were decapitated 4 weeks after delivery. Females from control group had administered 0.5 ml 0.9% NaCl intraperitoneally.

After decapitation, left kidneys were collected for histological evaluation. From collected kidneys histological preparations were performed for electron microscopy.

Systolic blood pressure was taken from animals on 10th, 20th day of pregnancy and 10 days and 4 weeks after delivery. In urine from 24 hour collection on 19^th day of pregnancy and 27 days after delivery protein concentration was determined.

RESULTS

Picture of kidney of animals from experimental group was significantly different from picture of kidney of controlled animals. Observed changes were segmental and focal.

In convoluted tubules of I and II degree were observed complete indistinctness of barriers between epithelial cells of tubular wall (Fig. 1, 4). Flattened and completely destroyed epithelial cells were observed in tubular lumen (Fig. 4). In some cells were observed "empty" cytoplasm without cell´s organelles (Fig. 1). In tubular lumen were present homogenous casts, naked nuclei, mitochondria and single tubular epithelial cells (Fig. 2, 3, 4). Focally in convoluted tubules of I degree was also destroyed brush border (Fig. 4). Epithelial basement membrane was focally thickened (Fig. 1, 3). In another places tubular epithelial cells were significantly swollen and tubular lumen was very narrow.

Fig. 1. Electronogram of part of renal convoluted tubule of female from experimental group. In epithelial cells is visible "empty" cytoplasm, destroyed connections between cells. Thickened basement membrane. Magn. 2 800x

Abbreviations: N - nucleus EC - tubular epithelial cell V - vacuole L - lumen of tubule BM - basement membrane

Fig. 2. Electronogram of part of renal convoluted tubule of female from experimental group. In tubular light are visible homogenous casts. Two nuclei in one tubular epithelial cell visible. Magn 2 800x

Abbreviations: N - nucleus EC - tubular epithelial cell V - vacuole L - lumen of tubule BM - basement membrane

Fig. 3. Electronogram of part of renal convoluted tubule of female from experimental group. In tubular light are visible homogenous casts. In epithelial cell pycnotic nucleus, and focally thickened basement membrane were visible. Magn. 2 450x

Abbreviations: N - nucleus EC - tubular epithelial cell V - vacuole L - lumen of tubule BM - basement membrane

Fig. 4. Electronogram of part of I degree renal convoluted tubule of female from experimental group. In tubular light are visible homogenous casts, naked nuclei, damaged mitochondria were observed. Focally destroyed brush border and cell membrane ruptured in tubular epithelial cell. In epithelial cell numerous vacuoles and damaged mitochondria were visible. Magn. 2 800x

Abbreviations: N - nucleus EC - tubular epithelial cell V - vacuole L - lumen of tubule BM - basement membrane

Systolic blood pressure assessed on 10^th day of pregnancy was statistically significantly higher in females from experimental group as compare to control group. On 20^th day of pregnancy it increased even more, and it slightly decreased 10 days after delivery. 4 weeks after delivery systolic blood pressure normalized to the range similar to control (Tab. 1). Similarly proteinuria in experimental group during pregnancy was significantly higher than after puerperium (Tab. 2).

Table 1. Values of systolic blood pressure

		Control group	Experimental group
pregnancy	10th day	76.0(+\-6.2)	100.9(+\-5.2)
	20 day	77.8(+\-9.4)	135.6(+\-4.8)
puerperium	10 day	80.5(+\-6.4)	120.0(+\-4.3)
	28 day(4 weeks)	82.0(+\-2.2)	89.0(+\-5.5)

Table 2. Protein concentration in urine (mg/24 hours)

		control group	Experimental group
pregnancy	19 day	8.00(+\-0.42)	221.34(+\-23.04)
puerperium	27day	7.75(+\-0.31)	132.26(+\-11.30)

DISCUSSION

In the literature could be find numerous reports about model of nephrotic syndrome in rat after single dose of adriamycin [2, 6, 11, 17, 18] or after other antibiotic from anthracyclin group with antineoplastic activity [5, 16]. Nephrotic syndrome developed in rat by adriamycin in dose 5 mg/kg of body weight was characterized by: proteinuria, hypoproteinemia with dysproteinemia, hipoalbuminemia, hypercholesterolemia and hypertriglicerydemia [2, 14, 17, 18,]. Described NS during pregnancy had features of preeclampsia [8, 10, 11, 13], with increased proteinuria comparatively to females with induced adriamycin NS without pregnancy [10]. Another typical for gestosis features include edemas of subcutaneous tissue, exudates to body cavities [7] swollen liver and kidneys and hypertension [1, 4, 9, 11, 12]. Hypertension described in literature was from 133.8 +/-4.8 mmHg (systolic pressure) [7] to 137+/-2.5mmHg [10]; in this study: from 110.9 +/-5.2mmHg (10^th day of pregnancy) to 135.6+/-4.8mmHg (20^th day of pregnancy)(Tab. Nr 1). In control group systolic pressure was about 78.8+/-10.3 [7], in this study: from 76.0 +/-6.2mmHg to82.0+/-2.2mmHg (Tab. 1). The experiment in present study was performed basing on numerous reports about model NS and preeclampsia after adriamycin [2, 8, 14, 18] and own research [6, 7].

The study was performed on female rats, which underwent pregnancy complicated with preeclampsia. They were investigated 4 weeks after delivery. Reports in the literature, and own study showed that factors (concentration of: total proteins, albumin, lipids, cholesterol, urine, creatinine) examined in that time in blood and urine of female rats showed the NS [7], but hypertension was not observed. Proteinuria and edemas although were significant, they came back to the level before pregnancy [7, 11].

In present experiment evident damage of renal tubular epithelial cells in females from experimental group was observed comparatively to corresponding cells from control group. Damages touched single cells or small group of cells. Many of cells showed pathological changes leading to nucleus and cytoplasm condensation. In the end degenerated cells died. Died cells or cellular elements coming from dying cells were secreted (into tubular lumen). In any tissue, cell death may follow in two distinct morphological and biochemical patterns: necrosis and apoptosis [3]. Apoptosis, opposite to necrosis touched single cells [15] and it is characterized with some signs. There were: plasmatic membrane damage, decrease of cell volume and nuclear pycnosis. Then nuclear fragmentation and creation of apoptotic bodies took place. The last step was phagocytosis of apoptotic bodies by neighbour cells. All processes went without inflammatory infiltration.

All that changes were visible in epithelial cells of proximal and distal convoluted tubules in rat females from experimental group. Changes in that cells had the features of apoptosis. Plasmatic membranes, brush border were destroyed, nuclei very often were picnotic and cytoplasm were without cellular organelles ("empty").

The histological changes described in present study were similar to that which appear in convoluted tubules rat females during pregnancy complicated with preeclampsia. However features of preeclampsia (hypertension, proteinuria, edemas) present during pregnancy disappeared or decreased starting from 10th day after delivery.

The trial to answer the question, why the features of preeclampsia decreased after puerperium in spite of the lack of any histological differences in convoluted tubules building of rat females after puerperium as compare to pregnant rat females with preeclampsia will be the subject of further quantities investigations. Helpful could be also the evaluation of kidney´s glomerulus.

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Agnieszka Pedrycz
Department of Histology and Embryology,
with Lab of Experimental Cytology,
Medical University of Lublin, Poland
Al. Racławickie 1, 20-059 Lublin, Poland


Marcin Wieczorski
Department of Histology and Embryology,
with Lab of Experimental Cytology,
Medical University of Lublin, Poland
Al. Racławickie 1, 20-059 Lublin, Poland

Zbigniew Boratyński
Deaprtment of Animal Anatomy and Histology,
Agricultural University of Lublin, Poland
Akademicka 12, 20-934 Lublin, Poland
phone: (+48 81) 445-60-33, 445-60-62
email: zbyszek.boratynski@ar.lublin.pl

Krystyna Czerny
Department of Histology and Embriology,
with Lab of Experimental Cytology,
Medical University of Lublin, Poland
Radziwiłłowska 11, 20-080 Lublin, Poland


Stanisław Flieger
Deaprtment of Animal Anatomy and Histology,
Agricultural University of Lublin, Poland
Akademicka 12, 20-950 Lublin, Poland
phone: (+48 81) 445-65-84

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