Renal Failure, 16(1), 109-116 (1994)

Roles of Hemodynamic and Tubular Factors in Gentamicin- Mediated Nephropathy

Akira Hishida, MD, Toshiaki Nakajima, MD, Masahito Yamada, MD, Akihiko Kato, MD, and Nishio Honda, MD

First Department of Medicine Hamamatsu University School of Medicine Hamamatsu, Japan

ABSTRACT

Gentamicin (GM) often causes polyuric acute renal failure (ARF) in hu- mans and animals. GM-mediated ARF in rats was accompanied with acti- vated renin-angiotensin system, increased renal endothelin content, and enhanced lipid peroxidation. Suppression of the renin-angiotensin activity by desoxycorticosterone acetate and saline drinking, and treatment with super- oxide dismutase attenuated the GM-induced decline in whole-kidney GFR with well-maintained RBF but did not reduce the severity of tubular necrosis. On the other hand, treatment with dimethylthiourea, a hydroxyl radical scav- enger, attenuated the GM-mediated decline in GFR and lessened tubular ne- crosis but did not ameliorate the reduction in RBF. These duta suggest contributions of both vascular and tubular factors to the GM-induced decline in GFR in rats. However, relative importance of these factors probably differs with different doses of the agent.

Key Words: Aminoglycoside; Hydroxyl radical; Renal blood flow; Renin-angiotensin systems; 'hbular necrosis.

Address correspondences to the authors at: 3600 Handa-cho, Hamamatsu, 431-31, Japan. Fax: Japan-53435 1626.

Copyright 0 1994 by Marcel Dekker, Inc.

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110

INTRODUCTION

Hishida et al.

Gentamicin (GM) often induces polyuric acute renal failure (ARF) in humans and an- imals. Controversy still exists, however, regarding the mechanisms for decline in the glomerular filtration rate (GFR) in GM-mediated ARE The administration of GM pro- vokes reduction in renal blood flow (RBF) and glomerular capillary ultrafiltration coef- ficient (K,) in rats (1-3). Improvements of RBF and K, caused by captopril, an angiotensin-converting enzyme inhibitor, result in the restoration of GFR toward the nor- mal level (1). Scanning electron microscopic observations disclosed reduction in the area of glomerular endothelial fenestrae in GM-mediated ARF in rats, a finding which sug- gests reduction in the glomerular filtering surface area and hence K, (4-6). An intrarenal infusion of angiotensin I1 also induces similar glomerular ultrastructural alterations (7), Based on these observations, the principal mechanisms for GM-mediated ARF have been thought to be a primary decline in GFR through reduction in RBF and K, due to renin- angiotensin stimulation. There is a controversy, however, concerning effects of capto- pril (8, 9)

On the other hand, GM produces tubular necrosis at the S1 and S2 segments of the proximal tubule and intratubular cast formation. Transmission electron microscopic ex- aminations revealed mitochondrial swelling, accumulation of “cytosegresome”- containing myeloid bodies, and loss of the brush border (1, 10, 11). A micropuncture study demonstrated that proximal tubular pressure increases in the early phase of GM- mediated ARF in rats, indicating a possible contribution of tubular obstruction to the GFR decline through a reduction in transcapillary hydraulic pressure difference (12, 13). Moreover, microinjection experiments showed that inulin injected into the proximal tu- bule leaks from the tubular lumen into the peritubular capillaries at a higher dose of GM (14) but not at a smaller dose (3). Recent evidence indicates that reactive oxygen me- tabolites are important mediators of ischemic and toxic renal cell injury. Indeed, the gen- eration of hydrogen peroxide has been found to be enhanced in GM-mediated ARF in rats (15). There is a possibility, therefore, that tubular injury due to enhanced generation of reactive oxygen metabolites also contributes to GFR decline in GM-mediated ARF through tubular obstruction and/or transtubular leakage of glomerular filtrate. However, a role of tubular injury in the GM-induced decline in GFR remains unclear.

This short review is focused on the roles of vascular and tubular factors in the GM- induced decline in GFR. Prior to our discussion, we summarize our published and un- published data concerning effects of renin-angiotensin suppression and free-radical scavenging.

EFFECTS OF RENIN-ANGIOTENSIN SUPPRESSION

Schor et al. (1) demonstrated that captopril attenuates GM-induced reduction in Kf and GFR. However, this agent acts not only as an angiotensin-converting enzyme inhib- itor but also as a free-radical scavenger (16). In our study (17), desoxycorticosterone ac- etate (DOCA) plus 1% saline drinking was used to suppress the renin-angiotensin activity. In this experiment, treatment with GM (40 mgkg of body wt/day) for 13 days induced significant reduction in RBF and the inulin clearance rate (CJ, and tubular ne- crosis in rats. Pretreatment with DOCA plus saline drinking for 5 weeks significantly

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Gentamicin-Mediated Nephropathy 111

*.O 1

Figure 1. Effects of DOCA plus saline and saline alone on RBF and Ci, in GM-treated rats. DOCA = desoxycorticosterone acetate, RBF = renal blood flow Ci, = inulin clearance, GM = gentarnicin. *p < .05 compared to control.

ameliorated the GM-induced reduction in RBF and Ci, (Fig. 1) but did not reduce the severity of tubular necrosis (Table 1). The GM-induced reduction in RBF was accompa- nied with reciprocal increase in the plasma renin activity (PRA) (3.3 k 0.6 in controls vs. 11.8 -t 1.6 ng AYmWh in GM-treated rats, p < .01). DOCA plus saline drinking significantly suppressed this GM-induced increase in PRA (0.9 4 0.3 ng AUmWh), and increased urine volume (UV) and urinary sodium excretion (U,,V) (Table 1). To eluci- date whether the beneficial effect of DOCA plus saline drinking results from renin-an- giotensin suppression or from well-maintained hydration and natriuresis, the effect of 1%

Table Z

Effects of Suppression of Renin-Angiotensin System on UV, UNaV and Tubular Necrosis in GM-Mediated Nephropathy

UV(mUday) N,,V (mEq/day) Necrotic tubulesa

Group 1: GM ~~ ~ -

16.8 * 2.8 0.7 +- 0.1 3.8 2 1.1

Group 2: (DOCA + GM) 53.3 * 13.1 10.6 2 1.4 2.5 2 0.3

Group 3: (saline + GM) 61.1 * 16.0 10.2 k 3.5 2.8 k 1 . 1

Group 1 vs. Group 2 p < .01 p < .01 NS

Group 1 vs. Group 3 p < .01 p < .05 NS

Note. Data are expressed as mean ? SEM. UV = urine volume, U,,V = urinary sodium excretion, GM = gentamicin, DOCA = desoxycorticosterone acetate. "Number of necrotic tubules per 1 high power field (X400).

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112 Hishida et al.

saline drinking alone on GM-mediated nephropathy was examined. Saline drinking alone resulted in significant increase in UV and UN,V but did not suppress PRA in GM-treated rats. Also, the GM-induced reduction in RBF and Cin was not attenuated by this proce- dure, indicating a possible beneficial effect of DOCA plus saline drinking not due to di- uresis and natriuresis. Furthermore, a significant inverse correlation was found between PRA and C, in GM-treated rats. It is likely, therefore, that GM-induced decline in GFR is mediated through impaired renal circulation due to renin-angiotensin stimulation.

EFFECTS OF FREERADICAL SCAVENGERS

Walker and Shah (15) noted that hydroxyl radical scavengers and iron chelators pre- vent the decline in Ci, and reduce the severity of tubular necrosis in GM-mediated ARF in rats, indicating a possible contribution of reactive oxygen metabolites to GM- mediated renal injury. However, the relation between the tubular damage and decline in GFR is obscure.

In our study (18), lipid peroxidation was significantly enhanced in GM-treated rats, supporting the previous data (15, 19). When dimethylthiourea (DMTU), a hydroxyl radical scavenger, was administered at a dose of 500 mglkglday just before the first in- jection of GM and then at 125 mgkg twice a day for 13 days, the GM-induced decline in Cin was attenuated significantly (Fig. 2). DMTU reduced the severity of tubular necrosis (Fig. 3). Furthermore, the increase in renal cortical lipid peroxidation product in GM- treated rats was completely inhibited by DMTU (the data are not shown). Thus hydroxyl radical would function as a mediator of GM-mediated tubular damage. On the other hand, DMTU had no effect on the GM-induced reduction in RBF. Also, the increase in

Figure 2. Effects of DMTU and SOD on Ci, and RBF in GM-treated rats. DMTU = dimethylthiourea, SOD = superoxide dismutase, RBF = rend blood flow, Ci, = inulin clear- ance, GM = gentamicin.

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Gentamicin-Mediated Nephropathy 113

-r

Figure 3. dirnethylthiourea, SOD = superoxide dismutase, GM = gentamicin.

Effects of DMTU and SOD on tubular necrosis in GM-treated rats. DMTU =

renal cortical renin and endothelin 1 contents following 13 days of treatment with GM was not significantly affected by DMTU (Fig. 4). Therefore, the beneficial effect of DMTU on GM-mediated ARF might be associated with lessened tubular injury but not with improved renal circulation.

In contrast with DMTU, recombinant human Cu/Zn SOD (30 mg/kg), which reduces superoxide anions, attenuated the GM-induced decline in Ci, with improved RBF (Fig. 2). The SOD treatment failed to lessen tubular necrosis (Fig. 3). Also, enhanced lipid peroxidation by GM was slightly but not significantly suppressed by SOD. Why does SOD improve RBF and hence GFR in GM-mediated ARF rats? SOD appears to have no remarkable direct vasodilating effect on kidney vessels, because this substance did not affect RBF in normal rats. Recent studies (20, 21) demonstrated that superoxide anions inactivate endothelial-derived relaxing factor (EDRF). Furthermore, in our experiment, SOD significantly lessened the GM-induced increase in renin and endothelin 1 contents in the renal cortex (Fig. 4). Taken together, these data suggest that the beneficial effect of SOD on GM-mediated ARF is mediated through well-maintained RBF due to altered intrarenal concentrations of these vasoactive substances.

ROLE OF VASCULAR AND TUBULAR FACTORS IN GM-MEDIATED ARF

As described above, DOCA plus saline drinking and SOD completely prevented the reduction in RBF and hence attenuated the decline in GFR in GM-treated rats, without

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114 Hishida et al.

T

T

Figure 4. treated rats. DMTU = dimethylthiourea, SOD = superoxide dismutase, GM = gentamicin.

Effects of DMTU and SOD on renal cortical renin and endothelin 1 contents in GM-

reducing the severity of tubular necrosis. Also, Schor et al. (1) have demonstrated that saline drinking significantly attenuates the GM-induced reduction in RBF, K,, and hence GFR but does not lessen tubular necrosis. These data suggest that renal hemodynamic alterations play a critical role in GM-induced decline in GFR. A possibility cannot be excluded, however, that additional factors also contribute to the GM-induced decline in GFR, since SOD partially protected against the GFR decline in rats despite well- maintained RBF above the control level. On the contrary, DMTU partially blunted the GM-induced decline in GFR without any change in RBF. Also, Baylis et al. (3) noted that 4 mgkglday of GM induces reduction in K, and hence GFR in the absence of a reduction in glomerular plasma flow. Furthermore, Fernandez-Repollet and Finn (22) showed that the normalization of RBF by acute volume expansion does not improve GFR in GM- treated rats. All of these data argue for the view that factors other than reduction in RBF and/or K, also contribute to the GM-induced decline in GFR. In contrast with DOCA plus saline drinking, DMTU lessened tubular necrosis. Thus the protective effect of DMTU against the GM-induced decline in GFR might be related to lessened tubular injury. In- deed, tubular obstructions due to casts (13) and back-leakage of filtrate (14) occur at a higher dose of GM but not at a small dose (3). It remains unclarified whether DMTU improves the GM-induced reduction in Kf.

The decline in GFR in GM-treated rats was associated with the increase in PRA and the renal cortical renin content. A close correlation between PRA and RBF in GM- treated rats suggests the important role of the renin-angiotensin system in the GM- induced reduction in RBF. Schor et al. (1) have demonstrated that captopril, an angiotensin-converting enzyme inhibitor, blunts effects of GM on single nephron GFR, glomerular plasma flow, and K,, and stressed the importance of renin-angiotensin stim- ulation in the development of GM nephrotoxicity, though the effect of captopril on GM-

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Gentamicin-Mediated Nephropathy 115

mediated ARF is controversial (8, 9). Also, the GM administration resulted in an increase in the renal cortical endothelin 1 content. Furthermore, some investigators dem- onstrated a contribution of thromboxane (23) and platelet-activating factor (2) to GM nephrotoxicity. The relative roles of these vasoactive substances in GM-mediated ARF remain unclarified.

From these observations, we conclude that both vascular and tubular factors contrib- ute to GM (40 mg/kg/day)-induced decline in GFR. The relative importance of these fac- tors probably differs with different doses of GM, with higher doses causing greater back- leakage of filtrate and/or more pronounced tubular obstruction (3, 13, 14, 24).

ACKNOWLEDGMENTS

This work was supported by a research grant sponsored by the Ministry of Education, Science, and Culture of Japan. The authors thank Schering-Plough for supplying gen- tamicin, Ube Industry for supplying SOD, and Takeda Chemical Industries for supplying the enzyme immunoassay kit for endothelin 1.

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