THE COMBINED EFFECT OF CYCLOSPORINE A AND GENTAMICIN ON ENZYMURIA IN THE SPRAGUE-DAWLEY RAT

Abstract

Male Sprague-Dawley rats (8 per group) were administered a single oral dose of cyclosporine A (10, 30 and 50 mg/day) for 5 days or vehicle (corn oil, 1.5 mL/kg) and urinary enzymes excretion was monitored. Only minor changes in enzymuria were observed in the 10 and 30 mg/kg group. However, in the 50 mg/kg group, nephrotoxicity was evident by significant increase in the excretion of N-acetyl-β-D-glucosaminidase (NAG), glutamate dehydrogenase (GDH), and lactate dehydrogenase (LDH on day 2 of treatment. As chemotherapeutic drug interaction with cyclosporine A (CyA) is thought to aggravate its nephrotoxicity, the effect of combined CyA (30 mg/kg) and the antibiotic gentamicin (50 mg/kg) for 5 days was investigated. Gentamicin alone caused a significant enzymuria, whilst co-treatment of rats with CyA gave rise to increased changes in enzymuria on days 1 and 2, between the groups receiving gentamicin + vehicle and those receiving CyA + gentamicin. This was particularly marked by significant changes in LDH excretion. In contrast these observed differences were not paralleled by changes in serum creatinine and other functional parameters. Treatment with gentamicin, appears to enhance CyA nephrotoxicity, but only in the first 2 days, after this there was no significant differences between the two groups. Our data suggest that urinary enzyme measurements could serve as a valuable non-invasive means of monitoring renal performance in animals or humans who may be exposed to combination of drugs. CyA is found not to potentiate the nephrotoxic effect of gentamicin in the animal model used in this study. It therefore appears safe to use the combined therapy particularly in the treatment of transplant patients.

• Cyclosporine A
• Gentamicin
• Enzymuria
• Renal toxicity

INTRODUCTION

Cyclosporine A (CyA) is undoubtedly the most effective immunosuppressive agent for use in the treatment of transplant recipients [1-3] and this has recently been extended to the management of patients with autoimmune disease [[4]]. However, the drug gives rise to toxic side effects, of which nephrotoxicity is one of the most important [[3]]. The characteristic abnormalities of which are defined functionally by diminished glomerular filtration rate (GFR), with elevated serum urea and creatinine [[5]] and structurally by atrophic tubular injury with interstitial fibrosis [[6]]. Although the aetiology of nephropathy is unknown, the effects of drug interaction have been implicated.

The incidence of CyA nephrotoxicity is reputed to be aggravated as a consequence of combined therapeutic or prophylactic treatment with other chemotherapeutic agents such as, antibiotic [[7]], diuretic [[8]], and oral contraceptives [[9]].

Aminoglycoside antibiotics, specifically gentamicin have been widely used to treat a broad-spectrum antibiotic in order to ameliorate opportunistic infections in the immunosuppressed CyA patients. Unfortunately high concentrations of gentamicin are nephrotoxic [10-11] and when combined with CyA in bone marrow transplant patients [12-13], or renal transplant cases [[7]], may lead to a severe deterioration in renal function.

At present it is not clear whether gentamicin and CyA act synergistically or whether the resultant CyA nephrotoxicity is simply additive due to the presence of the former agent, as inferred from early animal studies [[14]]. Clinically, nephrotoxicity is usually assessed by changes in clearance measurements, serum creatinine and or urea levels, but these tests are far too insensitive in the early detection of renal functional changes. Histopathological evaluations are very useful but these can be time consuming, as a routine investigation cannot be carried out on the same animal.

Studies conducted in our laboratory and by others have demonstrated the value of urinary enzymes as a sensitive and non-invasive test in the detection and monitoring of renal damage due to disease or nephrotoxic compounds [15-16]. In a recent study of rats receiving CyA at 40 mg/kg/day [[17]], cellular injury was clearly demonstrated by the marked increase of specific enzymes of subcellular origin into urine, which preceded changes in renal functional parameters.

In the present study a similar approach was adopted in which we evaluated the combined effects of CyA at a medial dose and gentamicin at an established toxic dose of 50 mg/kg [18-19] on enzymuria over a short time period. Although a large number of enzymes are excreted in urine [[16]], we have found only a few to be most suitable for diagnostic purposes. These enzymes are lactate dehydrogenase (LDH; EC 1.1.1.27), glutamate dehydrogenase (GDH; EC 1.4.1.2-4), alkaline phosphatase (ALP; 3.1.3.1), muramidase (MUR; EC 3.2.1.17) and N-acetyl-β-D-glucosaminidase (NAG; EC 3.2.1.30), and are specifically located in various regions of the kidney cells.

MATERIALS AND METHODS

Animals

Male Sprague-Dawley rats weighing 180–220g were purchased from Charles River (Margate, UK). All rats were acclimatised for 4 days to both the metabolism cages and the Perspex restraint cages (allows for the separation of urine from faeces) before the study began. The rats were kept at 23°C under a cycle of 12 h light/12 h dark and maintained on commercial rat chow with tap water ad libitum during the day, while access to water alone was provided during over night urine collection.

Reagents

Cyclosporine A (CyA) was donated by Sandoz Pharmaceuticals Ltd. (Feltham, U.K) and Gentamicin was purchased as a sterile aqueous solution of Gentamicin Sulphate (10 mg/mL) from, the Sigma Chemical Co.(Dorset, U.K). All other reagents were of analytical grade, prepared in distilled water re-distilled from an all glass still. The exception to this was CyA, which was prepared in a vehicle of corn oil.

Experimental Procedure

Following the acclimatisation period, control day (day 0) values were obtained.

In one set of experiment, rats were randomly divided into CyA treated groups and vehicle treated group (n = 8). Solutions of CyA were prepared in a vehicle of corn oil and given to rats by orogastric intubation. A volume of 0.15 mL per 100 g body weight was administered daily to groups of rats over a period of 5 days so as to give equivalent doses of 10, 30 and 50 mg/kg per day respectively. Control animals received vehicle only in the same volume.

In a second set of experiment, CyA at a dose of 30 mg/kg was administered in the morning (9.00 am) to a group of eight rats as described above and an identical number received the vehicle. In the evening (5.00 p.m.), gentamicin was given at a dose of 50 mg/kg to both groups, as a single subcutaneous injection for 5 days, followed by a recovery of 2 days.

Urine samples (16 h) were collected into test-tube maintained at 0°C on a day prior to treatment (day 0) and daily during treatment. Urine volume and the pH were routinely taken and the presence of blood, protein, glucose and ketone bodies was analysed semi-quantitatively using Labstix (Ames Laboratories Ltd. Stoke Poges, Bucks). The urine was centrifuged at 2000 g for 15 min. and the supernatant removed. A suitable aliquot was diluted with an appropriate volume of buffer to use for the enzyme assay as previously described to eliminate the effects of any inhibitors and activators [[20]].

Rats were anaesthetised with diethyl ether and blood was drawn from the cleaned tail tips. The blood was allowed to clot at 37°C and the serum separated following centrifugation (300 × g, 5 min). Blood aliquot from each rat was stored at −20°C until required.

Enzyme Assays, Protein, and Creatinine Determination

The activities of ALP, NAG, GDH, LDH and MUR were determined at 37°C under optimal conditions as previously described [[20]]. The activity of muramidase was expressed as g of cell walls hydrolysed per minute. The excretion of the enzymes into the urine was then expressed in terms of mU excreted per hour, and muramidase as μg/min per hour.

Protein was estimated by the Biuret method [[21]] using BSA standard.

The serum and urinary creatinine were determined using the appropriate kits purchased from the Sigma Chemicals Co. (Dorset, U.K).

Statistical Analysis

All data are shown as means ± SEM for at least two separate experiments. Normal ranges were established for all measurements before the various drug regimes, and the upper limit of normal (ULN) was defined as the mean value plus twice the standard deviation. Differences between treated animals and vehicle treated samples were evaluated statistically by using the one way analysis of variance (ANOVA) followed by a multiple comparison test, with statistical significance defined as P < 0.05.

RESULTS

Urinary Enzyme Changes With Increasing Doses of Cyclosporine A

The enzyme excretion of the different groups of rats is shown prior to any treatment, and from days 2 and 5 of CyA or the vehicle (Table 1). There were no significant increases in enzyme activity in the rats treated with vehicle alone, 10 mg/kg CyA or 30 mg/kg. However, in rats treated with 50 mg/kg CyA, there was a marked increase in the excretion of NAG, GDH and LDH on day 2 only. These increases were still present on day 4, but had returned to the pre-treatment values by day 5.

Table 1. Effect of Cyclosphorine A at Different Doses on Enzymuria

			Cyclosphorine A (mg/kg)
Enzyme	Day	Vehicle	10	30	50
	0	18.41 ± 4.05	21.18 ± 2.74	18.60 ± 2.86	21.32 ± 2.03
ALP	2	18.08 ± 8.09	17.60 ± 2.98	16.92 ± 2.63	21.84 ± 4.23
	5	12.48 ± 1.77	12.82 ± 2.25	17.48 ± 3.14	15.80 ± 9.20
	0	5.14 ± 1.14	7.10 ± 1.02	6.00 ± 1.07	5.72 ± 0.99
NAG	2	6.68 ± 0.73	6.52 ± 1.15	6.53 ± 1.50	8.91 ± 1.28^*
	5	5.77 ± 0.90	5.22 ± 0.91	6.72 ± 0.70	4.05 ± 0.95
	0	1.32 ± 0.23	1.45 ± 0.29	1.00 ± 0.25	1.31 ± 0.18
GDH	2	1.16 ± 0.23	1.87 ± 0.87	0.92 ± 0.18	3.50 ± 0.50^**
	5	1.50 ± 0.21	1.35 ± 0.26	1.51 ± 0.23	2.04 ± 0.56
	0	6.94 ± 1.72	7.60 ± 1.60	6.67 ± 1.98	6.78 ± 0.90
LDH	2	6.27 ± 1.61	8.67 ± 2.10	6.21 ± 0.94	21.73 ± 2.74^**
	5	6.37 ± 0.86	7.11 ± 0.90	9.11 ± 1.84	6.78 ± 2.02

Enzyme activity is expressed as mU/h and presented as mean ± SD for groups of 6–7 rats after various treatments.

^*P < 0.05;

^**P < 0.01 compared to pre-treatment (day 0) level, by Student's t-test.

Effect of a Single Dose of Gentamicin with Cyclosporine A on Enzyme Excretion

A single dose of gentamicin (50 mg/kg) alone caused significant increases of all enzymes examined. (Fig 1). Treatment with CyA at 30 mg/kg alone had no significant effect on enzyme excretion. However the combination of CyA and gentamicin led to a marked elevation in GDH, LDH and NAG excretion. There was a two-fold increase in the excretion of NAG and a five-fold elevation in LDH, whilst ALP remained unchanged.

Figure 1. The effect of a single dose of gentamicin (50 mg/kg) on enzymuria in vehicle and CyA (30 mg/kg/day) treated rats. Urine was collected 16 h overnight following the administration of the drugs. Results are mean ± SEM for groups of 6 rats. All values were compared to vehicle treatment by student's t-test for unpaired samples, ^*P < 0.05, ^**P < 0.01.

Effect of Concomitant Gentamicin Administration with Cyclosporine A on Enzymuria

The urinary excretion of ALP and GDH (Fig 2a and b) remained within normal limits for the duration of the experiment apart from a rise in GDH level to the ULN on day 7 (Fig 2b). There was no difference between the two groups of rats treated with the exception of ALP on day 6. On this occasion the group receiving gentamicin alone showed marked increase in the excretion of this enzyme than combined drug treatment group, although both sets of results were within the normal range.

Figure 2. Changes in a) ALP, b) GDH and c) NAG during administration of Gentamicin (50 mg/kg/day) to vehicle and CyA (30 mg/kg/day) treated rats. Results are mean ± SEM for groups of 6 rats. The upper limit of normal are represented as (- - - - - -). Open symbols denote Gentamicin + vehicle and the close symbols represent Gentamicin + CyA administered concomitantly. All values were compared to vehicle treatment by student's t-test for unpaired samples, ^*P < 0.05, ^**P < 0.01.

There was a significant elevation in the excretion of NAG in both groups on the first day and remained around the ULN for the duration of the experiment (Fig 2c).

However no significant distinction was apparent between the two groups.

The excretion of LDH by the group receiving CyA and gentamicin was substantially elevated during the first 3 days of gentamicin treatment, when compared with the group receiving the vehicle and gentamicin alone (Fig 3). But the excretion by the vehicle plus gentamicin treated group, rose steadily to form a peak by day 4. Thereafter, daily excretory fluctuations were observed in both groups, although the levels of excretions remained significantly high with only minor differences between the two groups for the rest of the experiment.

Figure 3. Changes in a) LDH and b) MUR during administration of Gentamicin (50 mg/kg/day) to vehicle and CyA (30 mg/kg/day) treated rats. Results are mean ± SEM for groups of 6 rats. The upper limit of normal are given as (- - - - - -). Open symbols denote Gentamicin + vehicle and the close symbols represent Gentamicin + CyA administered concomitantly. All values were compared to vehicle treatment by student's t-test for unpaired samples, ^*P < 0.05, ^**P < 0.01.

Muramidase excretion was initially raised significantly in both groups (Fig 3b). This returned to normal, and was abnormal again on day 7. As with the other results, there was no real difference between the gentamicin treated rats and those given gentamicin and CyA, in spite of the variation in the drug regimes.

Effect of Concomitant Gentamicin Administration with Cyclosporine A on Tubular Function

During gentamicin administration, urinary flow rate and creatinine excretion remained within normal limits for both treated groups. Although increases were later observed from day 5 following the withdrawal of gentamicin, both groups expressed this increase with no difference between them (Fig 4a and c). Changes in protein excretion of a similar magnitude were observed in both groups after gentamicin administration (Fig 4b). These rose initially then rapidly returned to levels within the pre-treatment range.

Figure 4. Changes in tubulo-glomerular renal function during administration of Gentamicin (50 mg/kg/day) to vehicle and CyA (30 mg/kg/day) treated rats. Results are mean ± SEM for groups of 6 rats. The upper limit of normal are given as (- - - - - -). Open symbols denote Gentamicin + vehicle and the close symbols represent Gentamicin + CyA administered concomitantly. All values were compared to vehicle treatment by student's t-test for unpaired samples, ^*P < 0.05, ^**P < 0.01.

Effect of Concomitant Gentamicin Administration with Cyclosporine A on Serum Creatinine

The serum creatinine was initially raised above normal in both groups without any marked differences (Fig 4d). Thereafter there was a decrease to within the normal range, which continued even after the withdrawal of gentamicin. Rats given the two drugs appeared to have a slightly high serum creatinine on the third day (Fig 4d) than that given gentamicin alone, but otherwise the two groups responded in the same way.

DISCUSSION

In the present study, the observation of broad changes in renal function after the administration of different doses of CyA was reflected by some early changes in enzymuria. This suggests that urinary enzyme measurements are as sensitive as histological studies, which have been used to establish a dose-related nephrotoxicity of CyA in rat [[22]].

However since high doses of CyA are required in the intact rat to elicit severe renal impairment, this suggest the interplay of other extra-renal factors in the clinical episodes of CyA nephrotoxicity. There is already a body of evidence to implicate the involvement of drug interactions [7-9].

The present study has considered the possible role the aminoglycoside gentamicin, as this agent is reported to enhance CyA toxicity in transplant patients [[7]], [12-13].

The administration of gentamicin and CyA separately induced different responses. The toxic dose of gentamicin (50 mg/kg), given as a single injection caused variable increases in all the enzymes which was consistent with early observation [18-19], in contrast to rats treated with CyA (30 mg/kg) when most of the enzyme excretion were normal. The two drugs are known to affect different portions of the nephron: gentamicin nephrotoxicity is associated with necrosis of the convoluted segment, whilst CyA predominantly causes vacuolation of the straight segment [[23]]. The administration of the two agents together might therefore be expected to cause an even more profound damage to the kidney.

In the periods which followed concomitant administration of the two drugs together over 5 days, tubular function appeared to be preserved as there were few changes in urinary flow rate and creatinine excretion. The changes in the excretion of ALP and GDH which remained within the normal range, suggests that the brush border membrane and mitochondria were unaffected. There were minor changes in the lysosomal enzyme (NAG) in both group of rats but these were small. However, when the rats given gentamicin and CyA were compared with those treated with gentamicin and vehicle, there was no real differences between the two groups for all three enzymes. These observations are surprising since gentamicin and CyA are reputed to effectively cause lysosomal and mitochondrial injury [[24]]. In contrast to this, the two drugs appeared to act synergistically when the excretion of LDH and MUR was examined.

However, the results were only transient and significant differences between the two groups were only seen in the period immediately at the start of the experiment.

The transient changes in enzymuria could possibly reflect an initial cellular sensitisation followed by gradual hypertrophy of the tubular cell [[25]]. The serum creatinine also rose in the initial phase of enzymuria but there was no significant differences between the two groups, and it gradually fell despite the continued administration of the drugs. This trend further supports the contention that serum creatinine is an insensitive index of progressive renal damage [[26]].

Some changes were also observed in all parameters following withdrawal of gentamicin, but these were quite mild with no real differences between the two groups. This phenomenon, which has also been observed in dogs treated with gentamicin [[27]], might be due to the residual effect of either drug in the blood. Early reports of gentamicin nephrotoxicity have been related to the peak and trough levels of the drug [[28]], and similar observations have been made with CyA [[29]].

Although we did not determine the serum CyA levels in the present study, the trends observed presently supports the contention of an increased serum CyA level following concomitant administration of gentamicin as inferred by early reports [[29]].

In the light of the present study it would appear that enhanced nephrotoxicity resulting from the combined therapy of gentamicin and CyA is only a transient phenomena as assessed by the excretion of urinary enzymes. Therefore gentamicin does not appear in the animal model used in this experiment to enhance the renal toxicity of CyA, even when given at a known toxic dose.

Thus provided careful monitoring of both drug levels is conducted, the degree of renal impairment can be reduced in transplant patients.