P.H. Whiting, Janet I. Duncan, M.P. Gavin, S.D. Heys,. J.G. Simpson, S.K. Asfar and A.W. Thomson ... I98I; Hamilton et al. I982). Moreover, drug-inducedstructural.
Br. J. exp. Path. (I985) 66, 535-542
Renal function in rats treated with cyclosporin following unilateral nephrectomy P.H. Whiting, Janet I. Duncan, M.P. Gavin, S.D. Heys, J.G. Simpson, S.K. Asfar and A.W. Thomson Departments of Pathology, Chemical Pathology and Surgery, University of Aberdeen, Aberdeen Received for publication
22
October I984
Summary. The effects of oral cyclosporin A (2 5 mg/kg/day) on renal function and structure in groups of normal, laparotomized and unilaterally nephrectomized DA rats was assessed over a 4-week period. In each group, the drug caused impairment of function, although there was evidence of improvement during the course of the study. Cyclosporin A toxicity was most marked in the nephrectomized animals, where histological examination at 4 weeks revealed extensive damage to proximal straight tubular cells. Only minimal structural damage was observed in the two other groups. Cyclosporin A concentrations in whole blood and kidney were estimated by radioimmunoassay at 4 weeks. Similar drug levels were found in normal, laparotomized and nephrectomized animals, except for an unexplained fall in blood levels in the laparotomy group. The significance of these observations is discussed in the context of current knowledge concerning the physiological effects, toxicology and pharmacology of cyclosporin A. Keywords: cyclosporin, kidney, nephrectomy, rat, toxicity, tubular cells
Considerable evidence has accumulated showing that the new immune suppressant cyclosporin A (CsA) (Sandimmun) which is of potential value in the control of allograft rejection, can impair renal function in man and laboratory animals (Calne et al. I978; Klintmalm et al. I 9 8 I; Lancet I 9 8 2; Thomson et al. I982). In patients with renal transplants, this untoward effect is characterized by elevations in serum urea and creatinine and by increases in urinary N-acetyl-fl-D-glucosaminidase (NAG) activity (Sweny et al. I98I) which indicate tubular cell damage. However, several authors have emphasized the difficulties in distinguishing CsA-induced nephrotoxicity from episodes of rejection (Keown et al. I98 I; Hamilton et al.
I982). Moreover, drug-induced structural changes similar to those seen in the kidneys of animals given 'high dosage' CsA (>25 mg/kg) have not been observed in patients. This apparent discrepancy remains to be resolved. Although several studies have centred on the tubulotoxicity of CsA in the surgically intact rat and in rats given renal allografts, the extent to which CsA-induced nephrotoxicity might be exacerbated by surgery and/or by nephrectomy has not been fully investigated. In this study, we have compared the effects of CsA treatment on renal function and structure in normal rats with those in rats following laparotomy or unilateral nephrectomy.
Correspondence: A.W. Thomson, Department of Pathology, University Medical Buildings, Foresterhill, Aberdeen AB9 2ZD. 535
536
P.H. Whiting et al. as I nmol 4-methylumbelliferone released per hour and enzyme levels were expressed five inbred male as units/mg creatinine.
Materials and methods Animals. Groups of four or DA rats, 220±30 g in body weight, purchased from Bantin and Kingman Ltd, Hull underwent nephrectomy on the left side whilst anaesthetized with chloral hydrate BP and diethyl ether. In sham-operated animals, a mid-line incision of the abdominal wall was performed without nephrectomy. Intact normal rats served as controls.
Cyclosporin. CsA (Sandoz Ltd, Basle, Switzerland) was dissolved initially at 20°C in anhydrous ethanol. A io% solution of the CsA-containing ethanol in olive oil BP was then prepared and O.I ml/ioo g administered to unanaesthetized rats by gastric intubation using a 4 fine-gauge intravenous cannula (Portex Ltd, Hythe, Kent, England). Experimental protocol. Groups of nephrectomized, sham-operated and control rats were given either CsA (25 mg/kg/day) commencing 24 h after surgery, or vehicle for 28 days. Analyses of serum and urine were conducted immediately before operation and at weekly intervals thereafter. On day 28, tail blood for CsA radioimmunoassay was collected into capillary containers (Sarstedt, UK) with EDTA (i mg/ml) as anticoagulant. The animals were then killed by terminal ether anaesthesia. Their kidneys were removed and bisected in the coronal plane. One half was fixed for microscopy and the other was weighed prior to CsA extraction.
Biochemistry. Blood samples (I.5 ml) were collected from cleaned tail tips under diethyl ether anaesthesia. Serum was stored at 20°C until assayed. Urine free of faecal contamination was collected overnight (I8 h) from rats placed in metabolic cages without food but with access to water throughout the collection period. Estimations of serum urea and creatinine and urinary N-acetyl-,BD-glucosaminidase (NAG) levels were conducted as described previously (Thomson et al. I 98 I). A unit of NAG activity was taken -
Microscopy. For light microscopy, kidney blocks were fixed in io% neutral buffered formalin and processed to acrylic resin: 2-pm sections were stained with haematoxylin and eosin. Histological assessment of renal tubular cell damage was performed 'blind' by an experienced renal histopathologist and the following scoring system was adopted to indicate the incidence of proximal straight tubular proffles affected: (o), normal; (i), < 5%; (2), > 5%; (3), > 50%; (4), > 90% abnormal. Tissue extraction of cyclosporin. CsA was extracted in ethanol (Atkinson et al. I983) at room temperature from homogenized half kidneys. The homogenates were washed three times and the pooled supernatants evaporated to io ml under an atmosphere of N2.
Radioimmunoassay ofcyclosporin. Estimations of CsA levels i 8 h after drug administration in whole blood samples (stored at - 20C and thawed once) and in kidney extracts were performed using radioimmunoassay kits (Sandoz Ltd, Basle) following the supplier's instructions. Statistics. The significance of differences between means was determined using Student's t-test for paired or unpaired samples as appropriate.
Results Serum and urine biochemistry The influence of CsA administered daily over 4 weeks to groups of normal, laparotomized and nephrectomized rats is shown in Fig. I. In all three groups of CsA-treated animals, there were two- to three-fold increases in serum urea within 7 days and elevated levels were maintained throughout the course of the study. Highest urea levels were observed
Cyclosporin toxicity after unilateral nephrectomy Normal
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Fig. i. Renal function in groups of normal, laparotomized and nephrectomized DA rats at weekly intervals after start of CsA treatment. 0 Vehicle-treated controls; 0 CsA. Results are means ± iSD. Symbols indicate significance of difference from day o (pretreatment) values. *P< O.OI; **P< O.OOI.
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538
Whiting et al.
Table i. Significance (P) of differences between groups of CsA-treated animals*
Day
Parameter Serum urea
Nephrectomy
vs
vs
vs
normal
normal
laparotomy
NS NS
NS NS
NS NS
