JOURNAL OF PATHOLOGY, VOL.
166: 297-30 1 (1 992)
CYCLOSPORIN REDUCES PROTEINURIA IN RATS WITH AMINONUCLEOSIDE NEPHROSIS KOH KOKUI, NORISHIGE YOSHIKAWA, HAJIME NAKAMURA AND HIROSHI ITOH
Department of Pediatrics, Kobe University School qf Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650, Japan Received 6 June 1991 Accepted 28 August 1991
SUMMARY The effect of cyclosporin (CS) was assessed in Sprague-Dawley rats with puromycin aminonucleoside (PA) nephrosis induced by a single intraperitoneal injection of PA. Three groups of rats were injected intraperitoneally with CS (10 mg/ kg body weight) daily, beginning 1 day before PA administration, o r 5 or 10 days after PA administration, for 10 days. CS significantly reduced proteinuria in rats with PA nephrosis in comparison with untreated nephrotic controls. After discontinuation of the CS treatment, proteinuria gradually increased, reaching values similar to those in control nephrotic rats. CS pretreatment did not prevent the induction of PA-induced nephrotic syndrome. Light microscopy and assessment of anionic sites in the glomerular basement membrane revealed no differences between normal rats, nephrotic controls, and CS-treated rats. These results show that CS can reduce proteinuria in PA nephrosis, but cannot ameliorate the glomerular changes. KEY
wom-Cyclosporin,
puromycin aminonucleoside nephrosis, proteinuria.
INTRODUCTION Recent clinical studies suggest that cyclosporin (CS) may have a beneficial effect in reducing proteinuria in minimal change nephrotic syndrome and nephrotic syndrome associated with focal segmental glomerulosclerosis (FSGS).'-3 Glomerular morphologicchanges in rats with puromycin aminonucleoside (PA) nephrosis resembling those in human minimal change nephrotic syndrome and FSGS have been d e m o n ~ t r a t ed .The ~ influence of CS on proteinuria has been studied extensively in many forms of immune-mediated experimental nephritis.5-" However, there is little published information about the effect of CS on proteinuria in nonimmunologically mediated types of experimental nephritis such as PA nephrosis." This study was designed with the following aims: (1) to establish whether CS reduces proteinuria and ameliorates glomerular injury in PA nephrosis, and (2) if so, to elucidate the mechanism of the phenomenon by studying the charge-selective function of the glomerular basement membrane (GBM). Addressee for correspondence: Dr Norishige Yoshikawa, Department of Pediatrics, Kobe University Hospital, 7-5- 1 Kusunoki-cho, Chuo-ku, Kobe 650, Japan.
0022-341 7/92/03029745 $05.00 Q
1992 by John Wiley & Sons, Ltd.
MATERIALS AND METHODS Preparation of animals Sixty male Sprague-Dawley rats (Shizuoka Laboratory Animals, Shizuoka, Japan), 7 weeks of age, were used. Fifty-two rats received a single intraperitoneal injection of puromycin aminonucleoside (Sigma Chemical Co., St. Louis, MO, U.S.A.) at 15 mg/100 g body weight. PA was dissolved in saline at a dilution of 20mg/ml. The other eight rats received an equal volume of saline (normal control group). CS solution for intravenous use was diluted 1: 10 with saline and administered intraperitoneally once a day for 10 days (10 mg/kg body weight per day). The following treatment schedules were employed and 13 animals were examined in each group: (a) CS application starting 1 day before administration of PA (group A); (b) CS application starting 5 days after administration of PA (group B); and (c) CS application starting 10 days after PA (group C). Thirteen animals received PA but no CS (nephrotic control rats). The animals were kept in metabolic cages and allowed free access to food and water. Daily 24-h urine was collected for measurement of protein by the sulphosalicylate method using a spectrophotometer (Bosch & Lomb,
298
K. KOKUI E T A L.
Germany). Serum for creatinine measurement was obtained at 10 and 20 days after PA administration. CS blood levels were measured at 5 days after the start of CS treatment in each CS-treated group. Total blood levels of CS were determined by radioimmunoassay in EDTA-treated whole blood samples.” All animals were anaesthetized with diethyl ether vapour and killed at 20 days after PA administration. The kidneys were then excised for light and electron microscopic study.
the mean number of anionic sites per l000nm length of the GBM.I6 Statistics Results were expressed as means & SD. Differences between groups were compared by the unpaired t-test or the Wilcoxon test at a significance level of P < 0.05. RESULTS
Preparation for light microscopy After killing, both kidneys were excised and one was cut into halves by a longitudinal section through the hilum, fixed in 10 per cent buffered formaldehyde by immersion, and embedded in paraffin. Sections approximately 4 pm thick were prepared and stained with periodic acid Schiff.
Proteinuria
Normal control values for 24-h urinary protein in the rats used in this study did not exceed 20mg. Figure 1 shows the 24-h urinary protein values for experimental rats following the injection of PA. Fifteen days after PA administration, the mean value for 24-h urinary protein excretion rose to 260567 mg/day in nephrotic control rats. Assessment of anionic sites in the GBM When CS treatment was initiated before PA Small renal tissue blocks were fixed in periodate- administration (group A), the proteinuria levels lysine-paraformaldehyde (PLP) at 4°C for &6 h, became significantly lower on days 5 and 15. Howwashed in increasing concentrations of sucrose in ever, proteinuria subsequently increased, reaching phosphate-buffered saline (PBS), and embedded values similar to those in the nephrotic controls by in Tissue-Tek compound (Lab-Tek Product).13 day 20. Frozen blocks were thawed and cut to dimensions In rats where CS treatment was started 5 days of less than 0.5mm’. The blocks were washed after PA administration (group B), the proteinuria three times with 0.1 M cacodylate buffer (pH levels on days 10 and 15 were significantly lower 7.4,400 mOsm/l) and immersed for 30 min in 0.5 per than those in nephrotic control rats. However, procent polyethyleneimine solution (PEI, MW 1200, teinuria subsequently increased, reaching values pH 7.4, 400 mOsm/l; Polyscience, PA, U.S.A.). similar to those in nephrotic controls by day 20. Then they were washed three times and stained by When CS treatment was started 10 days after PA immersion for 30 min in a mixture of 0.1 per cent administration (group C), the proteinuria levels glutaraldehyde and 1 per cent phosphotungstic acid on days 15 and 20 were significantly lower in (pH 7.4,400 mOsm/l). All procedures were carried comparison with nephrotic control rats. out in an ice-box. Specimens were post-fixed in 1 per Serum albumin levels on day 10 were significantly cent osmium tetroxide at 4°C for 1 h. They were lower in group A rats (2.4k0.3 g/dl), group B rats then dehydrated through a graded ethanol series (2.5 k 0.4 g/dl), group C rats (2.3 f0.5 g/dl), and and embedded in Quetol 812 resin. Ultrathin sec- nephrotic control rats (2.3 k 0 . 3 g/dl) than in tions were cut on a Reichert-Jung ultramicrotome, normal control rats (4.3 kO.1 g/dl) (P
166: 297-30 1 (1 992)
CYCLOSPORIN REDUCES PROTEINURIA IN RATS WITH AMINONUCLEOSIDE NEPHROSIS KOH KOKUI, NORISHIGE YOSHIKAWA, HAJIME NAKAMURA AND HIROSHI ITOH
Department of Pediatrics, Kobe University School qf Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650, Japan Received 6 June 1991 Accepted 28 August 1991
SUMMARY The effect of cyclosporin (CS) was assessed in Sprague-Dawley rats with puromycin aminonucleoside (PA) nephrosis induced by a single intraperitoneal injection of PA. Three groups of rats were injected intraperitoneally with CS (10 mg/ kg body weight) daily, beginning 1 day before PA administration, o r 5 or 10 days after PA administration, for 10 days. CS significantly reduced proteinuria in rats with PA nephrosis in comparison with untreated nephrotic controls. After discontinuation of the CS treatment, proteinuria gradually increased, reaching values similar to those in control nephrotic rats. CS pretreatment did not prevent the induction of PA-induced nephrotic syndrome. Light microscopy and assessment of anionic sites in the glomerular basement membrane revealed no differences between normal rats, nephrotic controls, and CS-treated rats. These results show that CS can reduce proteinuria in PA nephrosis, but cannot ameliorate the glomerular changes. KEY
wom-Cyclosporin,
puromycin aminonucleoside nephrosis, proteinuria.
INTRODUCTION Recent clinical studies suggest that cyclosporin (CS) may have a beneficial effect in reducing proteinuria in minimal change nephrotic syndrome and nephrotic syndrome associated with focal segmental glomerulosclerosis (FSGS).'-3 Glomerular morphologicchanges in rats with puromycin aminonucleoside (PA) nephrosis resembling those in human minimal change nephrotic syndrome and FSGS have been d e m o n ~ t r a t ed .The ~ influence of CS on proteinuria has been studied extensively in many forms of immune-mediated experimental nephritis.5-" However, there is little published information about the effect of CS on proteinuria in nonimmunologically mediated types of experimental nephritis such as PA nephrosis." This study was designed with the following aims: (1) to establish whether CS reduces proteinuria and ameliorates glomerular injury in PA nephrosis, and (2) if so, to elucidate the mechanism of the phenomenon by studying the charge-selective function of the glomerular basement membrane (GBM). Addressee for correspondence: Dr Norishige Yoshikawa, Department of Pediatrics, Kobe University Hospital, 7-5- 1 Kusunoki-cho, Chuo-ku, Kobe 650, Japan.
0022-341 7/92/03029745 $05.00 Q
1992 by John Wiley & Sons, Ltd.
MATERIALS AND METHODS Preparation of animals Sixty male Sprague-Dawley rats (Shizuoka Laboratory Animals, Shizuoka, Japan), 7 weeks of age, were used. Fifty-two rats received a single intraperitoneal injection of puromycin aminonucleoside (Sigma Chemical Co., St. Louis, MO, U.S.A.) at 15 mg/100 g body weight. PA was dissolved in saline at a dilution of 20mg/ml. The other eight rats received an equal volume of saline (normal control group). CS solution for intravenous use was diluted 1: 10 with saline and administered intraperitoneally once a day for 10 days (10 mg/kg body weight per day). The following treatment schedules were employed and 13 animals were examined in each group: (a) CS application starting 1 day before administration of PA (group A); (b) CS application starting 5 days after administration of PA (group B); and (c) CS application starting 10 days after PA (group C). Thirteen animals received PA but no CS (nephrotic control rats). The animals were kept in metabolic cages and allowed free access to food and water. Daily 24-h urine was collected for measurement of protein by the sulphosalicylate method using a spectrophotometer (Bosch & Lomb,
298
K. KOKUI E T A L.
Germany). Serum for creatinine measurement was obtained at 10 and 20 days after PA administration. CS blood levels were measured at 5 days after the start of CS treatment in each CS-treated group. Total blood levels of CS were determined by radioimmunoassay in EDTA-treated whole blood samples.” All animals were anaesthetized with diethyl ether vapour and killed at 20 days after PA administration. The kidneys were then excised for light and electron microscopic study.
the mean number of anionic sites per l000nm length of the GBM.I6 Statistics Results were expressed as means & SD. Differences between groups were compared by the unpaired t-test or the Wilcoxon test at a significance level of P < 0.05. RESULTS
Preparation for light microscopy After killing, both kidneys were excised and one was cut into halves by a longitudinal section through the hilum, fixed in 10 per cent buffered formaldehyde by immersion, and embedded in paraffin. Sections approximately 4 pm thick were prepared and stained with periodic acid Schiff.
Proteinuria
Normal control values for 24-h urinary protein in the rats used in this study did not exceed 20mg. Figure 1 shows the 24-h urinary protein values for experimental rats following the injection of PA. Fifteen days after PA administration, the mean value for 24-h urinary protein excretion rose to 260567 mg/day in nephrotic control rats. Assessment of anionic sites in the GBM When CS treatment was initiated before PA Small renal tissue blocks were fixed in periodate- administration (group A), the proteinuria levels lysine-paraformaldehyde (PLP) at 4°C for &6 h, became significantly lower on days 5 and 15. Howwashed in increasing concentrations of sucrose in ever, proteinuria subsequently increased, reaching phosphate-buffered saline (PBS), and embedded values similar to those in the nephrotic controls by in Tissue-Tek compound (Lab-Tek Product).13 day 20. Frozen blocks were thawed and cut to dimensions In rats where CS treatment was started 5 days of less than 0.5mm’. The blocks were washed after PA administration (group B), the proteinuria three times with 0.1 M cacodylate buffer (pH levels on days 10 and 15 were significantly lower 7.4,400 mOsm/l) and immersed for 30 min in 0.5 per than those in nephrotic control rats. However, procent polyethyleneimine solution (PEI, MW 1200, teinuria subsequently increased, reaching values pH 7.4, 400 mOsm/l; Polyscience, PA, U.S.A.). similar to those in nephrotic controls by day 20. Then they were washed three times and stained by When CS treatment was started 10 days after PA immersion for 30 min in a mixture of 0.1 per cent administration (group C), the proteinuria levels glutaraldehyde and 1 per cent phosphotungstic acid on days 15 and 20 were significantly lower in (pH 7.4,400 mOsm/l). All procedures were carried comparison with nephrotic control rats. out in an ice-box. Specimens were post-fixed in 1 per Serum albumin levels on day 10 were significantly cent osmium tetroxide at 4°C for 1 h. They were lower in group A rats (2.4k0.3 g/dl), group B rats then dehydrated through a graded ethanol series (2.5 k 0.4 g/dl), group C rats (2.3 f0.5 g/dl), and and embedded in Quetol 812 resin. Ultrathin sec- nephrotic control rats (2.3 k 0 . 3 g/dl) than in tions were cut on a Reichert-Jung ultramicrotome, normal control rats (4.3 kO.1 g/dl) (P
