International Urology and Nephrology 16 (3), pp. 243--251 (1984)

Effect of Some Simple Manoeuvres on the Course of Acute Renal Failure after Gentamycin Treatment in Rats J. HELLER Transplantation Research Centre of the Institute for Clinical a n d Experimental Medicine, Prague, Czechoslovakia (Received September 9, 1983)

For a period of 5 days, Wistar rats received Gentamycin (G), 100 mg/kg b.w./ day i.m. Three days after the last injection, the rats were sacrificed a n d the plasma concentrations of urea (Pu) and creatinine (Pc0 were determined. Both values were significantly higher than in the control rats receiving vehicle only. The increase was substantially greater in females than in males. The rats drinking isotonic NaC1 solution instead of water 7 days prior to G showed near n o r m a l Pu and Pcr values; drinking of N a H C O a had a similar protective effect. Isotonic sucrose solution was without any influence. The rats drinking Ca gluconate or NHaC1 solutions h a d similar or higher Pu a n d Pcr values as rats drinking water, but their body weight a n d overall condition markedly deteriorated. Brattleboro rats with diabetes insipidus exhibited a very similar course as Wistar rats; there was also no significant difference between the former a n d their heterozygous non-insipidic litter mates.

Introduction

The wide application of aminoglycoside antibiotics over the past ten years has been greatly impeded by their nephrotoxicity. Of these antibiotics Gentamycin (G) is the most toxic [19, 23]. The mechanism of this nephrotoxicity has been reviewed in a number of recent studies [4, 5, 13, 21]. Many studies deal also with the protection of the kidneys from G. Apart from the finding of a complete protective effect of diabetes mellitus [11, 24], the results of the studies are controversial (see Discussion). ~ The present study reviews some very simple methods of protection from G-induced nephrotoxicity on a rat model. The author chose deliberately a simple model and simple, practicable methods of protection (viz. hydration, urine alkalinization or acidification, extracellular fluid volume expansion, etc.). The purpose of the study was not to investigate the mechanisms capable of alleviating or aggravating G-induced nephrotoxicity. Methods

Most experiments were carried out on Wistar rats from our outbred colony, of either sex, weighing 180-200 g at entry into the experiment. Each group comprised 12 rats (6 males and 6 females) and were divided sex-wise into cages of 3 International~Urology and Ncphrology 16, 1984

244

Heller : Renal failure after 9entamycin

rats each. All were fed the standard VELAZ pellet diet. One group was composed of Brattleboro rats with congenital defect of vasopressin formation (DI rats); their heterozygous litter mates (HZ) served as controls. These rats were kindly supplied by Drs. J. Zicha and P. Klir of the Institute of Physiology, Czechoslovak Academy of Sciences, Prague. Because of their slower growth rate, DI rats were admitted to the experiments later than HZ. The experiments lasted 8 or 15 days. The body weight, food and liquid intake of all animals were measured daily. Day 0 was the control day; Gentamycin (100 mg/kg b.w./day) was applied intramuscularly on days 1 through 5; control rats received the same volume of vehicle. On day 8 the rats were decapitated and the freely flowing blood from the trunk was collected into test tubes with heparin. Blood plasma was analysed for urea [2] and creatinine [6]. Some experiments started a week earlier when the rats were already drinking the investigated solution; these days are denoted as - 7 through - 1. Further groups (of 12 rats each) were sacrificed on different days before and after the application of G so as to permit us to observe the course of changes of the plasma concentration of urea (Pu) and creatinine (Pcr)- The rats were subdivided into the following groups: C - controls receiving vehicle and drinking water; G1 - rats given G and drinking water; Groups Gz through G 8 receiving G and drinking isotonic solutions: - NaC1 62 G3 - sucrose G4 - NHaC1 G5 - NaHCO 3 Ca gluconate G 6 CDI - - control Brattleboro DI rats drinking water; C r l z - control Brattleboro non-insipidic heterozygotic rats; G m - Brattleboro insipidic rats receiving G; G H Z - Brattleboro non-insipidic heterozygotic rats given G. Statistical evaluation was performed by Student's t-test.

Results

Figures 1 and 2 show the course of Pu and PCr in rats drinking water and receiving G on day 1 through day 5. A clear sexual difference is seen. As the Pu and PCf are highest on day 8, this day was chosen for blood analysis. All data are shown in Table 1. As expected, a lower body weight increment was seen in control female Wistar rats (C) than in males. After G, this increment was lower in both sexes, yet the sexual difference was preserved. Food but not water intake was also lower after G. Pu and Pcr on day 8 were significantly higher than in C; a significant sexual difference was observed: the values in females were significantly higher Internati0nal Urology and Nephrology 16, 1984

245

Heller : Renal failure after 9entamycin Gentornyc n, 100 mg/kg b.w.i.m.

mmol/[

100

o

8O

8 o

60

o 0

8

o

o

40

o

,t

8o .o

I

I

I

I

I

I

I

[

I

I

0

1

2

3

t,

5

6

7

8

10

t

12

15

day

Fig. 1. The course of changes in plasma urea concentration (Pu)after Gentamycin in male (@) and female (O) Wistar rats

Gentamycin, 100 mglkg b.w.i.m.

jurnol / 1 500 400

o O

300

O

8 O

200 -

t

100 I

I

i

I

i

I

I

I

I

|

0

1

2

3

Z~

5

6

7

8

10

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12

i

15

day

Fig. 2. The course of changes in plasma creatinine concentration (Pcr) after Gentamycin in male (@) and female ( 9 Wistar rats

than in males. When isotonic saline replaced water as drinking liquid (Gs), the results were similar with exception of lower food and fluid intake and resulting body weight loss. However, when the rats drank isotonic saline 7 days before the start of G administration, a distinct protective effect was noted: Pv and Pcr were significantly lower than in group G 1 and Group Gz - subgroup without days - 7 International U r o l o g y and Nephr01ogy 16, 1984

Heller : Renal failure after gentamycin

246

t h r o u g h - 1. A l t h o u g h f o o d intake was lower, drinking liquid i n t a k e was higher in these rats. R a t s d r i n k i n g isotonic sucrose ( G ) h a d also a higher liquid intake, yet P u a n d Per were as high as in g r o u p G1. W i t h NH4C1 as d r i n k i n g fluid, a l o w er Table 1 Values on day 8 Group of rats

C

Drinking liquid Start of drinking (day)

Body weight ( ~ of starting value)

G1

water

water

0

0

c~

124.72 _ 9.82

107.87 12.78b

$

109.81 q- 8.88a

102.79 13.31a b

Food intake c~ 108.80 (mg/g b.w./day) -t- 11.12

Gz

G3

G,

NaC1

sucrose

NH4C1

0

--7

0

0

83.91 98.18 111.82 11.16b 10.17b 18.76b c 86.52 1 0 1 . 8 2 103.46 1 0 . 1 4 b 1 2 . 1 4 b 11.87a c b

82.03 9.18b c 85.52 11.02b c

83.31 10.02b c 93.17 10.08b

81.33 9.14b

41.13 8.18b

56.87 7.12b

72.72 10.12b

32.18 9.16a b

60.27 7.82b

63.88 9.66b

12.87 8.88b c

C

61.26 7.87a b

60.87 8.12b

45.27 9.17a b

63.18 8.28b

c

$

107.43 +__9 . 9 8

c

Liquid intake (#l/gb.w./day)

~

--7

c

154.71 --k 18.20

147.33 21.13

75.28 9.14b

241.63 38.17b

208.63 29.73b

73.87 12.12b

90.81 12.87b

C

C

C

C

C

168.12 q- 19.13

153.01 20.77

67.72 8.82b c

222.81 33.88b c

145.13 21.13a

38.88 18.23a d

118.12 27.27b c

C

Pu (mmolfi)

~

6.78 +0.17

28.22 3.07b

33.69 6.17b

20.00 3.19b

47.11 5.16b

39.16 9.72b

6.09 -t-0.61

73.67 9.18a b

72.67 8.47a b

C

C

19.17 2.88b c

54.83 8.16b

52.63 ll.18a b

49.13 10.16b

57.14 -t-4.87

178.18 22.13b

182.32 29.18b

88.83 9.98b

228.73 42.18b

199.26 32.13b

210.00 41.22b

61.33 q- 6 . 0 8

323.33 41.18a b

298.73 33.18a b

98.33 11.24b c

180.89 29.66b

187.16 33.18b

193.72 38.16b

C

P~ (mmol/1)

~

50.17 10.14b

C

Abbreviations: C through G D I - see Methods. a = significantly different males vs. females b = significantly different from corresponding control value International Urology and 1Nephrology 16, 1984

Heller : Renal failure after gentamycin

247

f o o d a n d fluid i n t a k e as w e l l as h i g h l y significant b l o o d loss w e r e o b s e r v e d . T h e u r i n a r y p H o f t h e s e rats was 5.98 +_ 0.64 as c o m p a r e d w i t h t h e v a l u e o f 7.03 _ 0.77 in g r o u p C a n d 7.12 _ 0.81; p < 0.01. I n m a l e s , P u b u t n o t P c r was h i g h e r o n

(mean + S.E.) G~ NaHCOs 0

Ge Ca gluconate --7

0

107.53 13.14b

111.83 14.18b

93.28a b c

--7

CHz water 0

GHZ water 0

CDI water 0

105.32 10.88b

107.40 13.52

GDI water 0

93.14 11.18b c 87.12 10.73b c

118.37 8.18

94.62a b c

84.72 10.27b c 91.89 12.18b c

108.22a

93.64 18.17a b

104.63 16.17

95.87 10.18b d 97.87 12.14b

68.72 10.16b

84.21 11.26b

39.92 8.78b

59.38 9.16b

84.82 9.16

66.81 7.71b

78.18 8.38

43.33 7.16b

c

C

48.81 7.77a b

53.72 8.28a b

62.28 8.88a b

35.16 7.18a b

80.13 7.14

51.78 7.08a b

81.43 9.98

51.29 8.46b

C

C

222.19 43.78b c 162.38 29.87a

276.48 39.12b c 218.37 33.62a b

53.87 9.22b c 78.82 9.02a b

104.71 14.26b c 70.81 10.06a b

256.72 67.87b d 263.22 55.14b d

C

C

C

13.22 3.27b

33.87 8.18b

41.67 9.27b

8.46 0.99

33.72 8.16b

7.89 0.89

46.58 9.27b

59.21 9.13a b

110.83 18.18a b

8.07 0.89

68.33 10.82a b

8.16 0.92

78.67a b

71.23 6.45 68.13 6.09

186.17 38.22b 361.00 68.12a b

76.83 8.28 71.26 8.02

195.50 28.36b 382.33 59.67a b

15.52 4.02b C

C

19.38 2.83b c

22.08 3.38a b

c

c

82.26b c 103.16b c

79.21b c 122.16a b c

137.29 10.14

154.45 12.43

1085.08 227.14

104.31 11.18

120.08 11.66

1028.36 286.44

C

205.64b 307.28a b

144.41 29.32b 420.82 87.16a b C

c---- significantly different from group G1 d = significantly different from group Gaz All differences at p < 0.01 International U r o l o g y a n d N e p h r o l o g y 16, 1984

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Heller : Renal failure after gentamycin

day 8 than in G 1 males. The results were similar irrespective of the time of the start of drinking NH~C1. After drinking NaHCO3 (Gs) significantly lower values of Pu and Pcr on day 8 were observed; these rats, too, had a higher fluid intake than both groups C and G1. The urinary pH of this group was 8.00 _+ 0.94; p < 0.001. The rats drinking Ca gluconate (G6) had a significant body weight loss, low food and fluid intake, and high Pu and Pcr. Very similar results as with Wistar rats were obtained in Brattleboro rats, both homo- and heterozygotic. After G, a decrease in body weight increment and food intake and an increase in Pc and Pcr were observed. The typical sexual difference (greater change in females than males) was also present. Homozygotic insipidic Brattleboro rats were characterized by known slow body weight gain and high water intake. After G, a decrease in body weight, food and water intake was seen. However, although depressed, the water intake was still significantly higher than in heterozygotes. Nevertheless, Pc and Pcr were both significantly increased to values not different from heterozygotes.

Discussion

Unlike Kahn et al. [18] who found no hyperazotaemia after a 4-day application of 1130mg/kg b.w./day of G to male Sprague-Dawley rats, our rats, both Wistar and Long-Evans-Brattlebor0, showed a significant rise of Pu and Pcr on day 8 after receiving the same dose for 5 days. Interestingly, the mate rats of both strains showed a much smaller rise than the female rats. To our knowledge, no similar finding has been reported in the literature nor can we explain it without further analysis. Isotonic NaC1 solution given as drinking liquid 7 days before the application of G had a distinctly protective effect which may be accounted for by several factors. Increased sodium intake might have been one of them. Compared with rats on a normal diet, rats receiving a high-salt diet showed a decreased G concentration in the renal cortex but no improvement of renal function and histology [3]. In contrast, a low-salt diet led not only to a higher concentration of G in the cortex tissue hut also to aggravated renal function and histology, and to a higher mortality rate. Chiu and Long [8] noted that i.v. administration of a bigger amount of isotonic NaC1 solution improved the course of G-induced acute renal failure in rats. It is speculative whether the effect of elevated sodium intake or one of the other possible causal factors, i.e. expansion of extracellular fluid volume (ECFV) is implicated in this event. Strangely enough, we observed the protective effect in our experiments only in rats which started to drink the NaC1 solution one week before (day - 7) the first application of G (day 1), on the other hand, those which received it only one day before (day 0) exhibited not only the same Pc and Pcr as group G1 but were showing also distinct weight losses. It seems therefore rational to assume that ECFV expanded only in rats which started to drink NaCI earlier, which, in turn, may be the sought mechanism of the effect. Neither can International Urology and Nephrology 16, 1984

Heller : Renal failure after #entamycin

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we rule out a further mechanism, i.e. accelerated tubular fluid flow rate starting already in the proximal convoluted tubules. Micropuncture studies have shown that expansion of ECFV by isotonic NaC1 solution results in a decrease of fluid reabsorption in proximal tubules [7, 10, 15]. The accelerated flow would help to wash out tubular debris observed after G by Neugarten et al. [20]. This conclusion is indirectly supported by the inefficacy of more distally acting diuretics in protection from G [1, 8] just as by our experiments with sucrose and DI rats. Rats starting to drink sucrose a week before G drank more than both C and G1 rats. It can be therefore assumed that they had a higher urine flow rate of the type of "water diuresis" which is characterized by a higher rate of tubular fluid flow in the diluting nephron segment. The same was true for insipidic rats which showed an identical course of failure as their non-insipidic heterozygotic litter mates. This last observation is at variance with what Texeira et al. had found [24], who noted a partial protection in their DI rats including a lower Pcr- The cause of this difference is not quite clear but may be due to a different experimental design. Unlike these authors who had administered 40 mg/kg/day G for 14 days, we used 100 mg/kg/day for 5 days. Soberon et al. [23] showed the overall daily dose to be a more important factor responsible for G-induced toxicity than the total dose. Summarizing, the protective effect of NaC1 drinking seems to depend on its inhibitory influence on proximal fluid reabsorption. Manoeuvres leading to suppression of more distal tubular reabsorption (sucrose, DI) seem to be ineffective. The role of urinary pH in G toxicity in rats is also obscure. Hsu et al. [16] observed that rats given G and drinking 19/0NH ~Ct solution had a higher Pu than those receiving tap water. Elliot et al. [12] made a similar observation. This is in accord with our experiments with male but not with female rats; however, both sexes exhibited strikingly lower body weight and their overall condition was distinctly poorer than in group G 1 rats. Elliot et al. [12] did not see any difference between the rats drinking NaHCO3 or water; Chiu et al. [9] reported a lower accumulation of G and somewhat improved course of G intoxication. We, on our part, noted a distinct protective effect of NaHCO3. However, since the intake of bicarbonate solution was higher by these rats than by rats of group G1, it can be assumed that in addition to urine alkalinization the beneficial effect may also be attributed to the elevated sodium intake or ECFV expansion similar to those seen after NaC1 solution. Recent reports describe a protective effect of elevated dietary Ca intake in ARF after G [14, 17, 22]. Our rats receiving Ca gluconate showed either the same Po and Pc~ (males) or higher (females) on day 8 as rats receiving water, their body weight was markedly lower and their overall physical condition poorer. We have no explanation for this difference as the scheme of G administration, the mode of Ca administration and the rat strains varied study by study. Whereas all the three quoted authors added CaCOa to the diet, our rats drank Ca gluconate solution. A more detailed analysis of the fate of Ca received is needed for clarifying the cause of observed differences. To summarize, drinking of isotonic NaCl and NaHCO3 solutions, particuInternational Urology and Nephrology 16, 1984

250

Heller : Renal failure after gentamyein

larly before the start of G a d m i n i s t r a t i o n , has a n obvious protective effect. I n contrast, m a n o e u v r e s resulting in "distal" diuresis (drinking sucrose, DI) were ineffective. NH~C1, causing u r i n e acidification, a n d Ca gluconate aggravated the c o n d i t i o n o f the rats. I n all cases a n d in the two strains, the course o f A R F after G was distinctly worse in female t h a n in male rats.

References 1. Adelman, R. D., Spangler, W. L., Beason, F., Ishikazi, G., Conzelman, G. M. : Furosemide enhancement of experimental gentamicin nephrotoxicity: Comparison of functional and morphological changes with activities of urinary enzymes. J. Infect. Dis., 140, 342 (1979). 2. Beale, R. N., Croft, D. : A sensitive method for the colorimetric determination of urea. J. Clin. Pathol. 14, 418 (1961). 3. Bennett, W. M., Hartnett, M. M., Gilbert, D., Houghton, D., Porter, G. A.: Effect of sodium intake on gentamicin nephrotoxicity in the rat. Proc. Soe. Exp. Biol. Med., 151, 736 (1976). 4. Bennett, W. M., Luft, F., Porter, G. A. : Pathogenesis of renal failure due to aminoglycosides and contrast media used in roentgenography. Am. J. Med., 69, 767 (1980). 5. Bennett, W. M. : Aminoglycoside nephrotoxicity: experimental and clinical considerations. Miner. Electrol. Metabol., 6, 277 (1981). 6. Bonsnes, R. W., Taussky, H.: On the colorimetric determination of creatinine by the Jaff6 reaction. J. Biol. Chem., 158, 581 (1945). 7. Brenner, B. M., Troy, J. L., Daugharty, T. M. : On the mechanism of inhibition in fluid reabsorption by the renal proximal tubule of the volume-expanded rat. J. Clin. Invest., 50, 1596 (1971). 8. Chiu, P. J. S., Long, J. F. : Effect of hydration on gentamicin excretion and renal accumulation in furosemide treated rats. Antimicrob. Agents Chemother., 14, 214 (1978). 9. Chiu, P. J. S., Miller, G. H., Long, J. F., Waitz, J. A. : Renal uptake and nephrotoxicity of gentamicin during urinary alkalinization in rats. Clin. Exp. Pharmaeol. Physiol., 6, 317 (1979). 10. Cortney, M. A., Mylle, M., Lassiter, W. E., Gottschalk, C. W. : Renal tubular transport of water, solute, and PAH in rats loaded with isotonic saline. Am. J. PhysioL, 209, 1199 (1965). 11. Cronin, R. E., Splinter, K. L., Ferguson, E. R., Henrich, W. L.: Gentamicin nephrotoxicity: protective effects of diabetes on cell injury. Miner. Electrol. Metabol., 9, 38 (1983). 12. Elliot, W. C., Parker, R. A., Houghton, D. C. : Effect of sodium bicarbonate and ammonium chloride ingestion in experimental gentamicin toxicity in rats. Res. Commun. Chem. Pathol. Pharmaeol., 28., 483 (1980). 13. Elliot, W. C., Houghton, D. C., Gilbert, D. N., Baines-Hunter, J., Bennett, W. M.: Gentamicin nephrotoxicity. J. Lab. Clin. Med., 100, 501 (1982). 14. Elliott, W. C., Gilbert, D. N., DeFehr, J., Bennett, W. M., McCarron, D. A. : Protection from gentamicin nephrotoxicity by dietary calcium loading. Kidney Int., 21, 216 (1982). 15. Heller, J., Nov~kov~, A.: Time course of changes of some renal parameters during infusion of isotonic saline solution in rats. Physiol. Bohemoslov., 17, 355 (1968). 16. Hsu, C. H., Kurtz, T. W., Easterling, R. E., Weller, J. M.: Potentiation of gentamicin nephrotoxicity by metabolic acidosis. Proe. Soe. Exp. Biol. IVied., 146, 894 (1974). IntcrnationalUrologyand Nephrology 16, 1984

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17. Humes, H. D., Sastrasinh, M., Weinberg, J. M.: A competitive inhibitor of gentamicin membrane receptor interaction which ameliorates G nephrotoxicity. Clin. Res., 30, 753A (1982). 18. Kahn, T., Bosch, J., Wiener, P., Dikman, S.: Course of gentamicin nephrotoxicity. Toxicology, 16, 49 (1980). 19. Luft, F. C., Bloch, R., Sloan, R. S., Yunn, M. N., Costello, R., Maxwell, D. R.: Comparative nephrotoxicity of aminoglycoside antibiotics in rats. J. lnfect. Dis., 138, 541 (1978). 20. Neugarten, J., Aynedjian, H. S., Bank, N. : The role of intratubular obstruction in gentamicin nephrotoxicity. Kidney Int., 21, 222 (1982). 21. Porter, G. A., Bennett, W. M.: Nephrotoxic acute renal failure due to common drugs. Am. J. Physiol., 241, F1 (1981). 22. Quarum, M., Houghton, D. C., Gilbert, D. N., McCarron, D. A., Bennett, W. M. : High calcium intake modifies experimental gentamicin nephrotoxicity. Clin. Res., 31, 76A (1983). 23. Soberon, L., Bowman, R. L., Pastoriza-Munos E., Kaloyanides, G. J.: Comparative nephrotoxicities of gentamicin, netilmicin, and tobramycin in the rat. J. Pharmacol. Exper. Ther., 210, 334 (1979). 24. Texeira, R. B., Kelley, J., Alpert, H., Pardo, V., Vaamonde, C. A.: Complete protection from gentamicin-induced acute renal failure in the diabetes mellitus rat. Kidney Int., 21, 600 (1982).

International Urology and Nephrology 16, 1984